Treatment of nervous system disorders using combinations of rxr agonists and thyroid hormones

ABSTRACT

Disclosed herein are methods of treating disease with a combination of a RXR agonist and a thyroid hormone.

RELATED APPLICATIONS

This application is a divisional of Ser. No. 16/118,001, filed Aug. 30,2018, which is continuation of U.S. patent application Ser. No.15/812,821, filed Nov. 14, 2017, which is a continuation of U.S. patentapplication Ser. No. 15/339,752 filed Oct. 31, 2016, now issued U.S.Pat. No. 9,877,941, which claims priority to U.S. Provisional PatentApplication No. 62/249,216, filed on Oct. 31, 2015. The entire contentof each of these applications is incorporated herein by reference.

FIELD

The present disclosure is directed to methods of treating nervous systemdisorders by inducing remyelination, neuroprotection, andimmunomodulation using a Retinoid X Receptor (RXR) agonist incombination with a thyroid hormone.

BACKGROUND

The current standard of care treatment for nervous system diseasesinclude several anti-inflammatory and immunomodulatory drugs thatpromote clinical benefit by modulating the patient's inflammatory/immuneresponses. While these therapies delay disease progression, they areunable to reverse the pathology or restore neurological function. Oneway to achieve significant advancement in the current standard of carefor nervous system disorder patients is to promote remyelination orneuroprotection, or both, and thereby regenerate or maintain healthyaxons and neurons.

SUMMARY

Disclosed herein are methods of treating nervous system disorders byinducing remyelination, neuroprotection, and immunomodulation using aRetinoid X Receptor (RXR) agonists in combination with a thyroid hormone

Specifically, disclosed herein are methods for treating a nervous systemdisorder, the methods comprising administering to an individual in needthereof a therapeutically effective amount of a RXR agonist and atherapeutically effective amount of a thyroid hormone, whereinadministration of the combination of the RXR agonist and the thyroidhormone treats the nervous system disorder in the individual moreeffectively than treatment with the RXR agonist or thyroid hormonealone. In some embodiments, the combination of RXR agonist and thyroxinetreats the nervous system disorder in the individual by both promotingremyelination and neuroprotection of neurons and modulating theindividual's immune system.

In some embodiments, the RXR agonist is a selective RXR agonist and is3,7-dimethyl-6(S),7(S)-methano,7-[1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphth-7-yl]2(E),4(E)heptadienoic acid, and has the structure of formula III:

In other embodiments, the RXR agonist is bexarotene or LG268.

In certain embodiments, the nervous system disorder is a central nervoussystem (CNS) disorder. In certain embodiments, the nervous systemdisorder is relapsing/remitting, primary progressive, and secondaryprogressive forms of multiple sclerosis (MS), diffuse white matterinjury in pre-term infants, neuromyelitis optica, acute disseminatedencephalomyelitis, Marburg multiple sclerosis, diffuse myelinoclasticsclerosis (Schilder's disease), Balo concentric sclerosis, solitarysclerosis, optic neuritis, transverse myelitis, amyotrophic lateralsclerosis (ALS), leukodystrophy (multiple variants, e.g.adrenoleukodystrophy, adrenomyeloneuropathy), Parkinson's disease,Alzheimer's disease, progressive supranuclear palsy, stroke, CNS traumaincluding traumatic brain injury and traumatic spinal cord injury,radiation induced neuroinflammation, radiation somnolence syndrome,Devic's disease, inflammatory demyelinating diseases, CNS neuropathies,central pontine myelinolysis, Tabes dorsalis (syphilitic myelopathy),progressive multifocal leukoencephalopathy, leukodystrophy, depression,schizophrenia, epilepsy, migraine, and dementias

In certain embodiments, the nervous system disorder is ademyelination-related disorder such as multiple sclerosis orradiation-induced nervous system inflammation.

In some embodiments, the demyelination-related disorder is a peripheralnervous system disorder such as Guillain-Barré Syndrome, acuteinflammatory demyelinating polyneuropathy, chronic inflammatorydemyelinating polyneuropathy, demyelinating diabetic neuropathy,progressive inflammatory neuropathy, drug- or toxin-induced neuropathy,such as chemotherapy-induced neuropathy or organophosphate-inducedneuropathy, anti-MAG peripheral neuropathy, Charcot-Marie-Tooth Disease,or copper deficiency.

In some embodiments, the therapeutically effective amount of the RXRagonist is about 0.001 mg/day to about 100 mg/day. In other embodiments,the therapeutically effective amount of the RXR agonist is about 1mg/day to about 20 mg/day. In some embodiments, the thyroid hormone isthyroxine. In certain embodiments, the therapeutically effective amountof thyroxine is about 12.5 μg/day to about 250 μg/day. In someembodiments, the RXR agonist is administered by nasal administration. Insome embodiments, both the RXR agonist and thyroxine are administered bynasal administration. In some embodiments, the RXR agonist isadministered orally. In some embodiments, the RXR agonist and thethyroxine are administered substantially simultaneously. In someembodiments, the RXR agonist and thyroxine are administered on differentschedules. In some embodiments, the thyroid hormone is administeredorally or subcutaneously.

In certain embodiments, treatment with the combination of RXR agonistand thyroxine reduces at least one symptom of a nervous system disorder,wherein the at least one symptom reduced is inflammation, fatigue,dizziness, headache, malaise, elevated fever and high body temperature,extreme sensitivity to cold in the hands and feet, weakness andstiffness in muscles and joints, weight changes, digestive orgastrointestinal problems, low or high blood pressure, irritability,anxiety, or depression, blurred or double vision, ataxia, clonus,dysarthria, fatigue, clumsiness, hand paralysis, hemiparesis, genitalanesthesia, incoordination, paresthesias, ocular paralysis, impairedmuscle coordination, weakness (muscle), loss of sensation, impairedvision, neurological symptoms, unsteady gait, spastic paraparesis,incontinence, hearing problems, or speech problems. In otherembodiments, treatment with the combination of RXR agonist and thyroxinereduces at least two symptoms of the nervous system disorder. In otherembodiments, treatment with the combination of RXR agonist and thyroxinereduces at least five symptoms of the nervous system disorder.

In some embodiments, the methods further comprise determining free serumthyroxine; and adjusting the dose of thyroxine to keep thyroxine levelsin an euthyroid range.

In some embodiments, further comprises administration of a neurotrophicfactor, or neurotrophic factor mimetic in combination with the RXRagonist and the thyroid hormone for treatment of a disease of thenervous system. In some embodiments, the neurotrophic factor is BDNF,GDNF, NGF, NT-3, bFGF, CNTF, NT-4/5, IGF, or insulin, or a mimeticthereof.

In certain embodiments, wherein the disease of the nervous system isParkinson's disease, Alzheimer's disease, a multiple sclerosis, an opticneuritis, a stroke, a CNS trauma, amyotrophic lateral sclerosis, aneuropathy, a nervous system hypoxia, a CNS toxicity, a dementia, aretinopathy, Huntington's disease, a synucleinopathy, epilepsy, autism,schizophrenia, depression, or an aging-related CNS degeneration.

In some embodiments, the neurotrophic factor is GDNF, or a GDNF mimetic,and the nervous system disease is Parkinson's disease. In someembodiments, the neurotrophic factor is GDNF, or a GDNF mimetic, and thenervous system disease is amyotrophic lateral sclerosis. In someembodiments, the neurotrophic factor is BDNF and the nervous systemdisease is Alzheimer's disease. In some embodiments, the neurotrophicfactor is insulin or insulin-like growth factor, and the nervous systemdisease is Alzheimer's disease. In some embodiments, the neurotrophicfactor is BDNF and the nervous system disease is multiple sclerosis. Insome embodiments, the neurotrophic factor is BDNF, and the nervoussystem disease is stroke, nervous system trauma, aging, or dementia. Insome embodiments, the neurotrophic factor is BDNF, GDNF, or insulin, andthe nervous system disease is aging-related nervous systemneurodegeneration. In certain embodiments, the neurotrophic factor ormimetic is administered by oral, parenteral, nasal, or topical routes,or by controlled release.

Also disclosed herein is the use of a combination of a RXR agonist, athyroid hormone, and a neurotrophic factor, or neurotrophic factormimetic, for in vitro promotion of survival or growth of neurons orglial cells, for subsequent implantation into the nervous system of apatient with a nervous system disorder.

Also disclosed herein are methods for promoting survival or repair ofneurons or glial cells in a patient with a nervous system disorder, themethod comprising administering to an individual in need thereof atherapeutically effective amount of a RXR agonist disclosed herein and atherapeutically effective amount of a thyroid hormone, whereinadministration of the combination of the RXR agonist and the thyroidhormone treats the nervous system disorder in the individual.

In certain embodiments, the method for promoting survival or repair ofneurons or glial cells further comprises administration of aneurotrophic factor, or neurotrophic factor mimetic to promote survivalor repair of neurons or glial cells in a patient with a nervous systemdisorder. In some embodiments, the neurotrophic factor is BDNF, GDNF,NGF, NT-3, bFGF, CNTF, NT-4/5, IGF, or insulin, or a mimetic thereof.

In some embodiments, the neurotrophic factor is GDNF, or a GDNF mimetic,and the nervous system disease is Parkinson's disease. In someembodiments, the neurotrophic factor is GDNF, or a GDNF mimetic, and thenervous system disease is amyotrophic lateral sclerosis. In someembodiments, the neurotrophic factor is BDNF and the nervous systemdisease is Alzheimer's disease. In some embodiments, the neurotrophicfactor is insulin or insulin-like growth factor, and the nervous systemdisease is Alzheimer's disease. In some embodiments, the neurotrophicfactor is BDNF and the nervous system disease is multiple sclerosis. Insome embodiments, the neurotrophic factor is BDNF, and the nervoussystem disease is stroke, nervous system trauma, aging, or dementia. Insome embodiments, the neurotrophic factor is BDNF, GDNF, or insulin, andthe nervous system disease is aging-related nervous systemneurodegeneration. In certain embodiments, the neurotrophic factor ormimetic is administered by oral, parenteral, nasal, or topical routes,or by controlled release.

Also disclosed herein are methods for treating multiple sclerosis, themethod comprising administering to an individual in need thereof atherapeutically effective amount of3,7-dimethyl-6(S),7(S)-methano,7-[1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphth-7-yl]2(E),4(E)heptadienoic acid (compound of Formula III) and a therapeuticallyeffective amount of thyroxine; and wherein administration of thecombination treats the multiple sclerosis in the individual moreeffectively than treatment with3,7-dimethyl-6(S),7(S)-methano,7-[1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphth-7-yl]2(E),4(E)heptadienoic acid or thyroid hormone alone.

Also disclosed herein are methods for treating a nervous systemdisorder, the method comprising administering to an individual in needthereof a therapeutically effective amount of3,7-dimethyl-6(S),7(S)-methano,7-[1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphth-7-yl]2(E),4(E)heptadienoic acid and a therapeutically acceptable amount of thyroxine;and wherein administration of the combination treats the nervous systemdisorder in the individual more effectively than treatment with3,7-dimethyl-6(S),7(S)-methano,7-[1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphth-7-yl]2(E),4(E)heptadienoic acid or thyroid hormone alone, and wherein the RXR agonistis delivered directly to the nervous system of the individual byintrathecal administration, epidural administration, cranial injectionor implant, or nasal administration.

Also disclosed are methods for treating Parkinson's disease, the methodcomprising administering to an individual in need thereof atherapeutically effective amount of3,7-dimethyl-6(S),7(S)-methano,7-[1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphth-7-yl]2(E),4(E)heptadienoic acid and a therapeutically effective amount of thyroxine;and wherein administration of the combination treats the Parkinson'sdisease in the individual more effectively than treatment with3,7-dimethyl-6(S),7(S)-methano,7-[1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphth-7-yl]2(E),4(E)heptadienoic acid or thyroid hormone alone.

Also disclosed herein are methods for treating Alzheimer's disease, themethod comprising administering to an individual in need thereof atherapeutically effective amount of 3,7-dimethyl-6(S),7(S)-methano,7-[1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphth-7-yl]2(E),4(E)heptadienoic acid and a therapeutically effective amount of thyroxine;and wherein administration of the combination treats the Alzheimer'sdisease in the individual more effectively than treatment with3,7-dimethyl-6(S),7(S)-methano,7-[1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphth-7-yl]2(E),4(E)heptadienoic acid or thyroid hormone alone.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows RXR agonist activation of transcription from RXRα, RXRβ,RXRγ, RARα, RARβ, and RARγ using transactivation assays.

FIG. 2 shows that RXR agonists combined with thyroid hormone attenuateexperimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice.

FIGS. 3A-B shows that RXR agonists reduce leukocyte infiltration intothe central nervous system. FIG. 3A depicts the number of CD4⁺ cells andFIG. 3B depicts the number of CD11c⁺ CD11b⁺ cells (myeloid DC) in micetreated with the selective RXR agonist IRX4204 (4204) versus the vehiclecontrol.

FIG. 4 shows RXR agonists attenuate EAE in SJL mice.

FIGS. 5A-D shows that IRX4204 selectively activates RXR-Nurr1heterodimers. Transactivation assay of IRX4204 (194204, Formula III) forfarnesoid X receptor FXR (FIG. 5A); for liver X receptors LXRα and LXRβ(FIG. 5B); for peroxisome proliferator-activated receptor PPARγ (FIG.5C); and for Nurr1 receptor in the presence or absence of RXR (FIG. 5D).

FIG. 6 shows the percentage of green fluorescent protein (EGFP) positiveoligodendrocytes after culture of oligodendrocyte precursor cellsderived from embryonic mouse brains with IRX4204 and thyroid hormone.

FIG. 7 depicts effects of selective RXR agonist IRX4204 on EAE in mice,

FIGS. 8A-B depicts expression of CCR6 (FIG. 8A) and CD49d (FIG. 8B) onsplenocytes from EAE mice treated with 200 μg/day of IRX4204 or control.

FIGS. 9A-D depicts quantification (FIG. 9A) and frequency (FIG. 9B) ofCD4+CD25hi cells, total number of effector and memory CD4 T cells (FIG.9C), and total number of activated CD4 T cells (FIG. 9D) in splenocytesfrom EAE mice treated with 200 μg/day of IRX4204 or control.

FIG. 10 depicts the total number of infiltrating CD4 T cells in the CNSof EAE mice treated with 200 μg/day of IRX4204 or control.

FIGS. 11A-D depicts re-stimulation of the infiltrating lymphocytes ofFIG. 10 to determine expression of interferon gamma (IFNγ) (FIG. 11A),IL-17A (FIG. 11B), tumor necrosis factor (TNF) (FIG. 11C), and IL-4(FIG. 11D).

FIGS. 12A-C depicts the quantification of co-expression of IFNγ andIL-17A by CD4 T cells of FIG. 10 expressing IL-17A and not IFNγ (FIG.12A), IL-17A and IFNγ (FIG. 12B), IFNγ and not IL-17A (FIG. 12C).

FIG. 13 depicts changes in paw placement behavior in a rat6-OHDA-induced model of Parkinson's disease upon treatment withcompounds and combinations described herein (*P<0.05 vs. vehicle usingone way ANOVA followed by Dunnett test).

FIG. 14 depicts the percent and fold change of EGFP+oligodendrocytesfollowing treatment of oligodendrocytes with IRX4204, thyroid hormone,and Vitamin D (*: P<0.05, student's t-test against DMSO control; Errorbar, SD).

FIGS. 15A-C depicts the percent change of EGFP+oligodendrocytesfollowing treatment of oligodendrocytes with IRX4204 and thyroid hormone(FIG. 15A: 10 nM IRX4204;

FIG. 15B: 1 nM IRX4204; FIG. 15C: 0.1 nM IRX4204). *** P<0.0001; **P<0.01.

FIGS. 16A-B depicts the effect of IRX4204 on remyelination in acuprizone-induced demyelination model. FIG. 16A depicts remyelination inthe hippocampus and FIG. 16B depicts remyelination in the cortex.

FIGS. 17A-B depicts quantitation of the size of myelinated axons. Thesize of myelinated axons after 6 weeks of treatment were quantified byImage J. Histogram of axon size distribution demonstrates a shift indistribution to larger axon diameter in IRX4204-treated axons (FIG.17A). Examination of the 3rd quartile date of axons about 0.7 μmdemonstrates a significant increase (P<0.0001) in the size of axons inthe upper quartile (FIG. 17B).

FIG. 18 depicts terminal circulating serum T4 levels in animals thatreceived vehicle, IRX4204, or IRX4204 and T4 (** P<0.005 vs vehicle andnaïve control).

FIG. 19 depicts a quantification of SM132 positive ovoids in corpuscallosum in animals that received vehicle, IRX4204, or IRX4204 and T4for 6 weeks (* P<0.05 vs Veh+Veh Control).

FIGS. 20A-C depicts a quantification of myelination of the corpuscallosum following in vivo treatment with combinations described herein,and a separation of the data into potential responders andnon-responders (one way ANOVA with Tukey's multiple comparisons, *P<0.05** P<0.01, **** P<0.001). FIG. 20A depicts the myelinated axons per CCunit; FIG. 20B depicts the density of myelinated axons (per 10,000 μm²);and FIG. 20C depicts the density of SM132+ovoids (per 250,000 μm²).

DETAILED DESCRIPTION

Many diseases of the nervous system are associated with demyelination ofaxons and neurons. Such disorders of demyelination may be autoimmunediseases or disorders of other etiologies. Multiple sclerosis (MS) is anexample of an autoimmune disorder which is also associated withdemyelination. Accordingly, an optimal drug or combination of drugs forthe treatment of MS would address the autoimmune aspects of the diseasewhile concurrently enhancing remyelination and providing neuroprotectionby preventing demyelination. MS is currently treated by severalimmunomodulatory drugs that provide clinical benefit by modulatingpatient immune responses and producing anti-inflammatory effects. Thesedrugs delay disease progression but do not reverse disease pathology orrestore neurological function by restoring myelination of damagedneurons. IRX4204 (194204, Formula III), a retinoid X receptor (RXR)ligand that has an unique mechanism of action in being a selectiveactivator of RXR homodimers and RXR-Nurr1 heterodimers, when combinedwith a thyroid hormone simultaneously provides immunomodulatoryactivities and also promotes remyelination and neuroprotection. IRX4204promotes the differentiation of suppressive Treg cells whilesimultaneously inhibiting the differentiation of pro-inflammatory Th17cells thereby favorably affecting the aberrantly skewed Th17/Treg cellratio which underlies human autoimmune diseases such as MS (seeco-pending US 2015/0038585, which is incorporated by reference for allit discloses regarding RXR agonists). Thus, by virtue of its effects onTh17/Treg cell ratios, IRX4204 will have clinical benefits similar tocurrent standard of care treatments in MS. In addition, IRX4204 promotesremyelination of demyelinated neurons and neuroprotection by preventingdemyelination. IRX4204 in combination with a thyroid hormone canadditionally promote further remyelination of demyelinated neurons andafford greater neuroprotection by preventing demyelination moreeffectively. Accordingly, a thyroid hormone and IRX4204, and other RXRligands of the same receptor activating profile, combination ofcompounds that provide both immunomodulatory activity and promoteremyelination and neuroprotection (and regeneration), will not onlydelay disease progression in MS but also effect neural maintenance andrepair by protecting and regenerating healthy axons and neurons. IRX4204together with thyroid hormone is expected to be an optimal drugcombination for the treatment of MS and other autoimmune diseases whichare also associated with demyelination.

The Retinoic Acid Receptors (RARs) and RXRs and their cognate ligandsfunction by distinct mechanisms. The RARs always form heterodimers withRXRs and these RAR/RXR heterodimers bind to specific response elementsin the promoter regions of target genes. The binding of RAR agonists tothe RAR receptor of the heterodimer results in activation oftranscription of target genes leading to retinoid effects. On the otherhand, RXR agonists do not activate RAR/RXR heterodimers. RXR heterodimercomplexes like RAR/RXR can be referred to as non-permissive RXRheterodimers as activation of transcription due to ligand-binding occursonly at the non-RXR protein (e.g., RAR); activation of transcription dueto ligand binding does not occur at the RXR. RXRs also interact withnuclear receptors other than RARs and RXR agonists may elicit some ofits biological effects by binding to such RXR/receptor complexes. TheseRXR/receptor complexes can be referred to as permissive RXR heterodimersas activation of transcription due to ligand-binding could occur at theRXR, the other receptor, or both receptors. Examples of permissive RXRheterodimers include, without limitation, peroxisome proliferatoractivated receptor/RXR (PPAR/RXR), farnesyl X receptor/RXR (FXR/RXR),nuclear receptor related-1 protein (Nurr1/RXR) and liver X receptor/RXR(LXR/RXR). Alternately, RXRs may form RXR/RXR homodimers which can beactivated by RXR agonists leading to rexinoid effects. Also, RXRsinteract with proteins other than nuclear receptors and ligand bindingto an RXR within such protein complexes can also lead to rexinoideffects. Due to these differences in mechanisms of action, RXR agonistsand RAR agonists elicit distinct biological outcomes and even in theinstances where they mediate similar biological effects, they do so bydifferent mechanisms. Moreover, the unwanted side effects of retinoids,such as pro-inflammatory responses or mucocutaneous toxicity, aremediated by activation of one or more of the RAR receptor subtypes.Stated another way, biological effects mediated via RXR pathways wouldnot induce pro-inflammatory responses, and thus, would not result inunwanted side effects.

Thus, aspects of the present specification provide, in part, a RXRagonist. As used herein, the term RXR agonist” refers to a compound thatbinds to one or more RXR receptors like an RXRα, a RXRβ, or a RXRγ in amanner that elicits gene transcription via an RXR response element. Asuse the term “selective RXR agonist” refers to the discriminatoryactivation of heterodimeric partners for RXR (such as a PPAR or a LXR)upon binding of a RXR agonist to one type of RXR heterodimer but not toanother.

In one embodiment, the selective RXR agonist does not activate to anyappreciable degree the permissive heterodimers PPAR/RXR, FXR/RXR, andLXR/RXR. In another embodiment, the RXR agonist activates the permissiveheterodimer Nurr1/RXR, but not other permissive RXR heterodimers. Oneexample of such a selective RXR agonist is3,7-dimethyl-6(S),7(S)-methano,7-[1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphth-7-yl]2(E),4(E)heptadienoic acid (IRX4204, 194204) disclosed herein, the structure ofwhich is shown in Formula III. In other aspects of this embodiment, theRXR agonist activates the permissive heterodimers PPAR/RXR, FXR/RXR, orLXR/RXR by 1% or less, 2% or less, 3% or less, 4% or less, 5% or less,6% or less, 7% or less, 8% or less, 9% or less, or 10% or less relativeto the ability of an activating RXR agonist to activate the samepermissive heterodimer. Examples of an RXR agonist which activates oneor more of PPAR/RXR, FXR/RXR, or LXR/RXR include, e.g., LGD1069(bexarotene) and LGD268.

IRX4204, like some other RXR ligands, does not activate non-permissiveheterodimers such as RAR/RXR. However, IRX4204 is unique in that itspecifically activates the Nurr1/RXR heterodimer and does not activateother permissive RXR heterodimers such as PPAR/RXR, FXR/RXR, andLXR/RXR. Other RXR ligands generally activate these permissive RXRheterodimers. Thus, all RXR ligands cannot be classified as belonging toone class. IRX4204 belongs to a unique class of RXR ligands whichspecifically and selectively activate RXR homodimers and only one of thepermissive RXR heterodimers, namely the Nurr1/RXR heterodimer. Thisunique receptor profile enables IRX4204 to have both immunomodulatoryand neural repair properties.

RXR agonists are known to suppress thyroid function. Treatment of humansubjects with the specific RXR agonist IRX4204 results first in areduction in plasma levels of TSH followed by a reduction in circulatingthyroxine levels. If a patient on IRX4204 develops adverse clinicalsymptoms due to the functional hypothyroidism, such clinical symptomscan be resolved by treatment of the patient with pharmacological dosesof thyroxine. However supplementation of RXR agonist therapy withthyroid hormones has not been utilized therapeutically. Surprisingly,the combination of a RXR agonist and a thyroid hormone producesunexpectedly better efficacy than the use of a RXR agonist alone,demonstrating synergism between the RXR agonist and thyroid hormone intreatment of the nervous system disorder independent of regulation ofplasma thyroid hormone levels.

Thus, the use of selective RXR homodimer, Nurr1/RXR activators, such asIRX4204, together with thyroid hormone provides uniquely effective waysof treating nervous system disorders.

Binding specificity is the ability of a RXR agonist to discriminatebetween a RXR receptor and a receptor that does not contain its bindingsite, such as, e.g., a RAR receptor. Certain RXR agonists can activateRXR homodimers as well as most permissive RXR heterodimers (for example,RXR/PPAR, RXR/LXR, RXR/Nurr1); such RXR agonists are known asnon-selective RXR agonists. Certain other RXR agonists activate RXRhomodimers and, unexpectedly, activate only one or a few RXRheterodimers. Such RXR agonists (e.g., IRX4204) are known as selectiveRXR agonists.

Thus, disclosed herein are selective RXR agonists having the structureof formula I:

where R⁴ is lower alkyl of 1 to 6 carbons; B is —COOH or —COOR⁸ where R⁸is lower alkyl of 1 to 6 carbons, and the configuration about thecyclopropane ring is cis, and the configuration about the double bondsin the pentadienoic acid or ester chain attached to the cyclopropanering is trans in each of the double bonds, or a pharmaceuticallyacceptable salt of the compound.

In an exemplary embodiment, a selective RXR agonist is a compound havingthe structure of formula II:

wherein R is H or lower alkyl of 1 to 6 carbons.

In a further exemplary embodiment, a selective RXR agonist is3,7-dimethyl-6(S),7(S)-methano,7-[1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphth-7-yl]2(E),4(E)heptadienoic acid (IRX4204), and has the structure of formula III:

In certain embodiments, the ester form of3,7-dimethyl-6(S),7(S)-methano,7-[1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphth-7-yl]2(E),4(E)heptadienoic acid is not within the scope of the present disclosure.

In certain embodiments, a non-selective RXR agonist is bexarotene(TARGRETIN®,4-[1-(3,5,5,8,8-pentamethyl-6,7-dihydronaphthalen-2-yl)ethenyl]benzoicacid).

In other embodiments, a RXR agonist is LG268 (LG100268,2-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)cyclopropyl]pyridine-5-carboxylicacid).

As used herein, the term “thyroid hormone” refers to thyroxine andtriiodothyronine. Thyroxine (thyroid hormone T₄, levothyroxine sodium)is a tyrosine-based hormone produced by the thyroid gland and isprimarily responsible for regulation of metabolism. Thyroxine is aprohormone for triiodothyronine (T₃). RXR agonists are known to suppressthyroid function. The clinical symptoms associated with thishypothyroidism can be treated with thyroid hormone. Howeversupplementation of RXR agonist therapy with thyroid hormones to increaseefficacy of the RXR agonist has not been utilized therapeutically.

As disclosed herein, the combination of a RXR agonist and a thyroidhormone increases myelination in the central or peripheral nervoussystem or provides neuroprotection by preventing demyelination by atleast about 10% to at least about 25%, at least about 10% to at leastabout 50%, at least about 10% to at least about 75%, at least about 10%to at least about 100%, at least about 10% to at least about 200%, atleast about 10% to at least about 300%, at least about 10% to at leastabout 400%, at least about 10% to at least about 500%, at least about25% to at least about 50%, at least about 25% to at least about 75%, atleast about 25% to at least about 100%, at least about 25% to at leastabout 200%, at least about 25% to at least about 300%, at least about25% to at least about 400%, at least about 25% to at least about 500%,at least about 50% to at least about 100%, at least about 50% to atleast about 200%, at least about 50% to at least about 300%, at leastabout 50% to at least about 400%, or at least about 50% to at leastabout 500%, relative to myelination levels in the absence of treatmentwith the RXR agonist and thyroid hormone.

In yet other aspects of this embodiment, the combination of a RXRagonist and a thyroid hormone increases differentiation ofoligodendrocyte progenitor cells into functional oligodendrocytes in thecentral or peripheral nervous system by at least about 10% to at leastabout 25%, at least about 10% to at least about 50%, at least about 10%to at least about 75%, at least about 10% to at least about 100%, atleast about 10% to at least about 200%, at least about 10% to at leastabout 300%, at least about 10% to at least about 400%, at least about10% to at least about 500%, at least about 25% to at least about 50%, atleast about 25% to at least about 75%, at least about 25% to at leastabout 100%, at least about 25% to at least about 200%, at least about25% to at least about 300%, at least about 25% to at least about 400%,at least about 25% to at least about 500%, at least about 50% to atleast about 100%, at least about 50% to at least about 200%, at leastabout 50% to at least about 300%, at least about 50% to at least about400%, or at least about 50% to at least about 500%, relative todifferentiation levels in the absence of treatment with the RXR agonistand thyroid hormone.

In yet another aspect of the present specification, the combination of aRXR agonist and a thyroid hormone increases the rate of myelin repair byat least about 10% to at least about 25%, at least about 10% to at leastabout 50%, at least about 10% to at least about 75%, at least about 10%to at least about 100%, at least about 10% to at least about 200%, atleast about 10% to at least about 300%, at least about 10% to at leastabout 400%, at least about 10% to at least about 500%, at least about25% to at least about 50%, at least about 25% to at least about 75%, atleast about 25% to at least about 100%, at least about 25% to at leastabout 200%, at least about 25% to at least about 300%, at least about25% to at least about 400%, at least about 25% to at least about 500%,at least about 50% to at least about 100%, at least about 50% to atleast about 200%, at least about 50% to at least about 300%, at leastabout 50% to at least about 400%, or at least about 50% to at leastabout 500%, relative to myelin repair rates in the absence of treatmentwith the RXR agonist and thyroid hormone.

Aspects of the present specification provide, in part, a compositioncomprising a RXR agonist and a thyroid hormone. Exemplary RXR agonistsare IRX4204, bexarotene, and LG268. Also provided are methods oftreating nervous system disorders with a combination of IRX4204 andthyroxine.

Aspects of the present disclosure provide, in part, treatment of anervous system disorder, such as a demyelination-related disorder. Ademyelination-related disorder is any disease or disorder of the nervoussystem in which the myelin sheath of neurons is damaged. This damageimpairs the conduction of signals in the affected nerves. In turn, thereduction in conduction ability causes deficiency in sensation,movement, cognition, or other functions depending on which nerves areinvolved. Both the central nervous system and the peripheral nervoussystem can be involved.

Some demyelination-related disorders are caused by genetics, some byinfectious agents or toxins, some by autoimmune reactions, some byradiation injury, and some by unknown factors. Neuroleptics can alsocause demyelination. The precise mechanism of demyelination is notclearly understood but there is substantial evidence that the body's ownimmune system is at least partially responsible, causingdemyelination-related disorders to be considered autoimmune disorders.

Autoimmune disorders, including some nervous system and demyelinationdisorders, arise from an overactive immune response of the body againstsubstances and tissues normally present in the body resulting in a breakin tolerance toward self-antigens. In other words, the body actuallyattacks its own cells because the immune system mistakes some part ofthe body as a pathogen and attacks it. Characterized by the developmentof pathogenic T cell populations infiltrating the target organ ortissue, autoimmune disorders may be restricted to certain organs orinvolve a particular tissue in different places.

Nervous system disorders can be broadly divided into central andperipheral nervous system disorders, depending on the organs mostaffected. Central nervous system disorders include, without limitation,relapsing/remitting, primary progressive, and secondary progressiveforms of multiple sclerosis (MS), diffuse white matter injury inpre-term infants, neuromyelitis optica, acute disseminatedencephalomyelitis, Marburg multiple sclerosis, diffuse myelinoclasticsclerosis (Schilder's disease), Balo concentric sclerosis, solitarysclerosis, optic neuritis, transverse myelitis, amyotrophic lateralsclerosis (ALS), leukodystrophy (multiple variants, e.g.adrenoleukodystrophy, adrenomyeloneuropathy), Parkinson's disease,Alzheimer's disease, progressive supranuclear palsy, stroke, traumaticCNS injury including brain and spinal cord trauma, radiation inducedneuroinflammation, radiation somnolence syndrome, Devic's disease,inflammatory demyelinating diseases, CNS neuropathies like thoseproduced by vitamin B12 deficiency, central pontine myelinolysis,myelopathies like Tabes dorsalis (syphilitic myelopathy),leukoencephalopathies like progressive multifocal leukoencephalopathy,radiation induced central nervous system inflammation andleukodystrophies. Peripheral nervous system disorders include, withoutlimitation, Guillain-Barre Syndrome, acute inflammatory demyelinatingpolyneuropathy, chronic inflammatory demyelinating polyneuropathy,demyelinating diabetic neuropathy, progressive inflammatory neuropathy,drug- or toxin-induced neuropathy, such as chemotherapy-inducedneuropathy or radiation-induced neuropathy or organophosphate-inducedneuropathy, anti-MAG peripheral neuropathy, Charcot-Marie-Tooth Disease,radiation induced neuropathy, copper deficiency, depression,schizophrenia, epilepsy, migraine, and dementias.

In some embodiments, the disorder is not cachexia.

In certain embodiments, the nervous system disorder is Alzheimer'sdisease. In other embodiments, the disorder is not Alzheimer's disease.

In one embodiment, the demyelination-related disorder is MS. Multiplesclerosis is currently treated by several immunomodulatory drugs thatprovide clinical benefit by modulating patient immune responses andproducing anti-inflammatory effects. These drugs delay diseaseprogression but do not prevent disease progression by preventingdemyelination and affording neuroprotection or reverse disease pathologyor restore neurological function by restoring myelination of damagedneurons. The selective RXR agonist IRX4204 has a unique mechanism ofaction in that it is a specific activator of RXR homodimers andRXR/Nurr1 heterodimers and simultaneously provides immunomodulatoryactivities and promotes remyelination and prevents demyelinationparticularly when used in combination with thyroid hormone. IRX4204promotes the differentiation of suppressive Treg cells whilesimultaneously inhibiting the differentiation of pro-inflammatory Th17cells, thereby favorably affecting the aberrantly skewed Th17/Treg cellratio which underlies human autoimmune diseases such as MS. Thus, byvirtue of its effects on Th17/Treg cell ratios, the combination ofIRX4204 and a thyroid hormone is expected to have clinical benefitssimilar to, or better than, current standard of care treatments in MS.Moreover, IRX4204 in combination with thyroid hormone is more effectivein promoting remyelination of demyelinated neurons and affordingneuroprotection by preventing demyelination. Accordingly, thecombination of IRX4204 and a thyroid hormone will not only delay diseaseprogression in MS but also effect neural repair by regenerating healthyaxons and neurons.

Aspects of the present disclosure includes, in part, reducing at leastone symptom associated with a nervous system disorder. The actualsymptoms associated with a nervous system disorder disclosed herein arewell known and can be determined by a person of ordinary skill in theart by taking into account factors, including, without limitation, thelocation of the nervous system disorder, the cause of the nervous systemdisorder, the severity of the nervous system disorder, the tissue ororgan affected by the nervous system, and the nervous system associatedwith inflammation. Non-limiting examples of symptoms reduced by a methodof treating a nervous system disorder disclosed herein includeinflammation, fatigue, dizziness, headache, malaise, elevated fever andhigh body temperature, extreme sensitivity to cold in the hands andfeet, weakness and stiffness in muscles and joints, weight changes,digestive or gastrointestinal problems, low or high blood pressure,irritability, anxiety, depression, blurred or double vision, ataxia,clonus, dysarthria, clumsiness, hand paralysis, hemiparesis, genitalanaesthesia, incoordination, paresthesias, ocular paralysis, impairedmuscle coordination, weakness (muscle), loss of sensation, impairedvision, neurological symptoms, unsteady gait, spastic paraparesis,incontinence, hearing problems, and speech problems. In certainembodiments, treatment with a combination of an RXR agonist and athyroid hormone reduces at least one symptom, at least two symptoms, atleast three symptoms, at least four symptoms, or at least five symptomsof a nervous system disorder.

In certain embodiments, the RXR agonist treats MS and reduces one ormore symptoms of MS such as, but not limited to, pain in the back oreyes, tremors, muscle cramping, difficulty walking, inability to rapidlychange motions, involuntary movements, muscle paralysis, musclerigidity, muscle weakness, problems with coordination, stiff muscles,clumsiness, muscle spasms, overactive reflexes, fatigue, dizziness, heatintolerance, poor balance, vertigo, weakness, excessive urination atnight, leaking of urine, persistent urge to urinate, urinary retention,pins and needles, abnormality of taste, uncomfortable tingling andburning, blurred vision, double vision, vision loss, erectiledysfunction, sexual dysfunction, anxiety, mood swings, slurred speech,impaired voice, acute episodes, constipation, depression, difficultyswallowing, difficulty thinking and understanding, headache, heavy legs,numbness, numbness of face, rapid involuntary eye movement, sleepdeprivation, tongue numbness, or difficulty raising the foot.

Efficacy of a compound or combination disclosed herein in MS can bedetermined by improvement in one or more recognized scales of MSincluding, but not limited to, the Expanded Disability Status Scale(EDSS; Kurtzke scale), Functional System Score (FSS), MS Progression:Disease Steps (DS), and MS Progression: Multiple Sclerosis FunctionalComposite (MSFC).

In certain embodiments, the RXR agonist treats Parkinson's disease andreduces one or more symptoms of Parkinson's disease such as, but notlimited to, tremor (can occur at rest, in the hands, limbs, or can bepostural): stiff muscles, difficulty standing, difficulty walking,difficulty with bodily movements, involuntary movements, musclerigidity, problems with coordination, rhythmic muscle contractions, slowbodily movement, slow shuffling gait, daytime sleepiness, earlyawakening, nightmares, restless sleep, fatigue, dizziness, poor balance,restlessness, amnesia, confusion in the evening hours, dementia,difficulty thinking and understanding, impaired voice, soft speech,voice box spasms, anxiety, apathy, distorted sense of smell, loss ofsmell, dribbling of urine, leaking of urine, jaw stiffness, reducedfacial expression, blank stare, constipation, depression, difficultyswallowing, drooling, falling, fear of falling, loss in contrastsensitivity, neck tightness, small handwriting, trembling, orunintentional writhing.

Efficacy of a compound or combination disclosed herein in Parkinson'sdisease can be determined by improvement in one or more recognizedscales of Parkinson's disease including, but not limited to, MultipleSclerosis Functional Composite (MSFC), Unified Parkinson's DiseaseRating Scale (UPDRS), the Hoehn and Yahr scale, and the Schwab andEngland Activities of Daily Living Scale.

In certain embodiments, the RXR agonist treats Alzheimer's disease andreduces one or more symptoms of Alzheimer's disease such as, but notlimited to, mental decline, difficulty thinking and understanding,confusion in the evening hours, delusion, disorientation, forgetfulness,making things up, mental confusion, difficulty concentrating, inabilityto create new memories, inability to do simple math, inability torecognize common things, aggression, agitation, difficulty with selfcare, irritability, meaningless repetition of own words, personalitychanges, lack of restraint, wandering and getting lost, anger, apathy,general discontent, loneliness, mood swings, depression, hallucination,paranoia, loss of appetite, restlessness, inability to combine musclemovements, jumbled speech.

Efficacy of a compound or combination disclosed herein in Alzheimer'sdisease can be determined by improvement in one or more recognizedscales of Alzheimer's disease including, but not limited to, theDementia Severity Rating Scale (DSRS), Mini-Mental State Examination(MMSE), Alzheimer's Disease Assessment Scale (ADAS), including theADAS-cog, Neuropsychological Test Battery (NTB), Severe ImpairmentBattery (SIB), an Activities of Daily Living Scale, a Clinical GlobalImpression (CGI) scale, BEHAVE-AD, Brief Psychiatric Rating Scale(BPRS), Alzheimer Disease Related Quality of Life (ADRQL), DementiaQuality of Life Instrument (DQoL), the Quality of Life—Alzheimer'sDisease (QoL-AD), and the Quality of Life in Late-Stage Dementia Scale(QUALID). Also within the scope of this disclosure is the treatment ofother disorders with a combination of an RXR agonist and a thyroidhormone. Such disorders include cancer without limitation on the type ofcancer, autoimmune diseases, and muscular diseases.

Aspects of the methods of the present disclosure include, in part,treatment of a mammal. A mammal includes a human, and a human can be apatient. Other aspects of the present disclosure provide, in part, anindividual. An individual includes a mammal and a human, and a human canbe a patient.

A combination of a RXR agonist disclosed herein and a thyroid hormone,is generally administered to an individual as a pharmaceuticalcomposition. Pharmaceutical compositions may be prepared by combining atherapeutically effective amount of at least one RXR agonist and onethyroid hormone, or pharmaceutically acceptable acid addition saltsthereof, as an active ingredient, with conventional acceptablepharmaceutical excipients, and by preparation of unit dosage formssuitable for therapeutic use. As used herein, the term “pharmaceuticalcomposition” refers to a therapeutically effective concentration of anactive compound, such as, e.g., any of the compounds disclosed herein.Preferably, the pharmaceutical composition does not produce an adverse,allergic, or other untoward or unwanted reaction when administered to anindividual. A pharmaceutical composition disclosed herein is useful formedical and veterinary applications. A pharmaceutical composition may beadministered to an individual alone, or in combination with othersupplementary active compounds, agents, drugs or hormones. Thepharmaceutical compositions may be manufactured using any of a varietyof processes, including, without limitation, conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping, and lyophilizing. The pharmaceuticalcomposition can take any of a variety of forms including, withoutlimitation, a sterile solution, suspension, emulsion, lyophilizate,tablet, pill, pellet, capsule, powder, syrup, elixir, or any otherdosage form suitable for administration.

A pharmaceutical composition produced using the methods disclosed hereinmay be a liquid formulation, semi-solid formulation, or a solidformulation. A formulation disclosed herein can be produced in a mannerto form one phase, such as, e.g., an oil or a solid. Alternatively, aformulation disclosed herein can be produced in a manner to form twophase, such as, e.g., an emulsion. A pharmaceutical compositiondisclosed herein intended for such administration may be preparedaccording to any method known to the art for the manufacture ofpharmaceutical compositions.

Liquid formulations suitable for parenteral injection or for nasalsprays may comprise physiologically acceptable sterile aqueous ornonaqueous solutions, dispersions, suspensions or emulsions and sterilepowders for reconstitution into sterile injectable solutions ordispersions. Formulations suitable for nasal administration may comprisephysiologically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions. Examples of suitable aqueous andnonaqueous carriers, diluents, solvents or vehicles include water,ethanol, polyols (propylene glycol, polyethyleneglycol (PEG), glycerol,and the like), suitable mixtures thereof, vegetable oils (such as oliveoil) and injectable organic esters such as ethyl oleate. Proper fluiditycan be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersions and by the use of surfactants.

Pharmaceutical formulations suitable for administration by inhalationinclude fine particle dusts or mists, which may be generated by means ofvarious types of metered, dose pressurized aerosols, nebulizers, orinsufflators.

Semi-solid formulations suitable for topical administration include,without limitation, ointments, creams, salves, and gels. In such solidformulations, the active compound may be admixed with at least one inertcustomary excipient (or carrier) such as, a lipid and/or polyethyleneglycol.

Solid formulations suitable for oral administration include capsules,tablets, pills, powders and granules. In such solid formulations, theactive compound may be admixed with at least one inert customaryexcipient (or carrier) such as sodium citrate or dicalcium phosphate or(a) fillers or extenders, as for example, starches, lactose, sucrose,glucose, mannitol and silicic acid, (b) binders, as for example,carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone,sucrose and acacia, (c) humectants, as for example, glycerol, (d)disintegrating agents, as for example, agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain complex silicates andsodium carbonate, (e) solution retarders, as for example, paraffin, (f)absorption accelerators, as for example, quaternary ammonium compounds,(g) wetting agents, as for example, cetyl alcohol and glycerolmonostearate, (h) adsorbents, as for example, kaolin and bentonite, and(i) lubricants, as for example, talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate or mixturesthereof. In the case of capsules, tablets and pills, the dosage formsmay also comprise buffering agents.

In liquid and semi-solid formulations, a concentration of a RXR agonisttypically may be between about 50 mg/mL to about 1,000 mg/mL. In aspectsof this embodiment, a therapeutically effective amount of a therapeuticcompound disclosed herein may be from, e.g., about 50 mg/mL to about 100mg/mL, about 50 mg/mL to about 200 mg/mL, about 50 mg/mL to about 300mg/mL, about 50 mg/mL to about 400 mg/mL, about 50 mg/mL to about 500mg/mL, about 50 mg/mL to about 600 mg/mL, about 50 mg/mL to about 700mg/mL, about 50 mg/mL to about 800 mg/mL, about 50 mg/mL to about 900mg/mL, about 50 mg/mL to about 1,000 mg/mL, about 100 mg/mL to about 200mg/mL, about 100 mg/mL to about 300 mg/mL, about 100 mg/mL to about 400mg/mL, about 100 mg/mL to about 500 mg/mL, about 100 mg/mL to about 600mg/mL, about 100 mg/mL to about 700 mg/mL, about 100 mg/mL to about 800mg/mL, about 100 mg/mL to about 900 mg/mL, about 100 mg/mL to about1,000 mg/mL, about 200 mg/mL to about 300 mg/mL, about 200 mg/mL toabout 400 mg/mL, about 200 mg/mL to about 500 mg/mL, about 200 mg/mL toabout 600 mg/mL, about 200 mg/mL to about 700 mg/mL, about 200 mg/mL toabout 800 mg/mL, about 200 mg/mL to about 900 mg/mL, about 200 mg/mL toabout 1,000 mg/mL, about 300 mg/mL to about 400 mg/mL, about 300 mg/mLto about 500 mg/mL, about 300 mg/mL to about 600 mg/mL, about 300 mg/mLto about 700 mg/mL, about 300 mg/mL to about 800 mg/mL, about 300 mg/mLto about 900 mg/mL, about 300 mg/mL to about 1,000 mg/mL, about 400mg/mL to about 500 mg/mL, about 400 mg/mL to about 600 mg/mL, about 400mg/mL to about 700 mg/mL, about 400 mg/mL to about 800 mg/mL, about 400mg/mL to about 900 mg/mL, about 400 mg/mL to about 1,000 mg/mL, about500 mg/mL to about 600 mg/mL, about 500 mg/mL to about 700 mg/mL, about500 mg/mL to about 800 mg/mL, about 500 mg/mL to about 900 mg/mL, about500 mg/mL to about 1,000 mg/mL, about 600 mg/mL to about 700 mg/mL,about 600 mg/mL to about 800 mg/mL, about 600 mg/mL to about 900 mg/mL,or about 600 mg/mL to about 1,000 mg/mL.

In semi-solid and solid formulations, an amount of a RXR agonisttypically may be between about 0.01% to about 45% by weight. In aspectsof this embodiment, an amount of a therapeutic compound disclosed hereinmay be from, e.g., about 0.1% to about 45% by weight, about 0.1% toabout 40% by weight, about 0.1% to about 35% by weight, about 0.1% toabout 30% by weight, about 0.1% to about 25% by weight, about 0.1% toabout 20% by weight, about 0.1% to about 15% by weight, about 0.1% toabout 10% by weight, about 0.1% to about 5% by weight, about 1% to about45% by weight, about 1% to about 40% by weight, about 1% to about 35% byweight, about 1% to about 30% by weight, about 1% to about 25% byweight, about 1% to about 20% by weight, about 1% to about 15% byweight, about 1% to about 10% by weight, about 1% to about 5% by weight,about 5% to about 45% by weight, about 5% to about 40% by weight, about5% to about 35% by weight, about 5% to about 30% by weight, about 5% toabout 25% by weight, about 5% to about 20% by weight, about 5% to about15% by weight, about 5% to about 10% by weight, about 10% to about 45%by weight, about 10% to about 40% by weight, about 10% to about 35% byweight, about 10% to about 30% by weight, about 10% to about 25% byweight, about 10% to about 20% by weight, about 10% to about 15% byweight, about 15% to about 45% by weight, about 15% to about 40% byweight, about 15% to about 35% by weight, about 15% to about 30% byweight, about 15% to about 25% by weight, about 15% to about 20% byweight, about 20% to about 45% by weight, about 20% to about 40% byweight, about 20% to about 35% by weight, about 20% to about 30% byweight, about 20% to about 25% by weight, about 25% to about 45% byweight, about 25% to about 40% by weight, about 25% to about 35% byweight, or about 25% to about 30% by weight.

A pharmaceutical composition disclosed herein can optionally include apharmaceutically acceptable carrier that facilitates processing of anactive compound into pharmaceutically acceptable compositions. As usedherein, the term “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for contact withthe tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem complicationscommensurate with a reasonable benefit/risk ratio. As used herein, theterm “pharmacologically acceptable carrier” is synonymous with“pharmacological carrier” and refers to any carrier that hassubstantially no long term or permanent detrimental effect whenadministered and encompasses terms such as “pharmacologically acceptablevehicle, stabilizer, diluent, additive, auxiliary, or excipient.” Such acarrier generally is mixed with an active compound or permitted todilute or enclose the active compound and can be a solid, semi-solid, orliquid agent. It is understood that the active compounds can be solubleor can be delivered as a suspension in the desired carrier or diluent.Any of a variety of pharmaceutically acceptable carriers can be usedincluding, without limitation, aqueous media such as, e.g., water,saline, glycine, hyaluronic acid and the like; solid carriers such as,e.g., starch, magnesium stearate, mannitol, sodium saccharin, talcum,cellulose, glucose, sucrose, lactose, trehalose, magnesium carbonate,and the like; solvents; dispersion media; coatings; antibacterial andantifungal agents; isotonic and absorption delaying agents; or any otherinactive ingredient. Selection of a pharmacologically acceptable carriercan depend on the mode of administration. Except insofar as anypharmacologically acceptable carrier is incompatible with the activecompound, its use in pharmaceutically acceptable compositions iscontemplated. Non-limiting examples of specific uses of suchpharmaceutical carriers can be found in Pharmaceutical Dosage Forms andDrug Delivery Systems (Howard C. Ansel et al., eds., Lippincott Williams& Wilkins Publishers, 7^(th) ed. 1999); Remington: The Science andPractice of Pharmacy (Alfonso R. Gennaro ed., Lippincott, Williams &Wilkins, 20^(th) ed. 2000); Goodman & Gilman's The Pharmacological Basisof Therapeutics (Joel G. Hardman et al., eds., McGraw-Hill Professional,10^(th) ed. 2001); and Handbook of Pharmaceutical Excipients (Raymond C.Rowe et al., APhA Publications, 4^(th) edition 2003). These protocolsare routine and any modifications are well within the scope of oneskilled in the art and from the teaching herein.

A pharmaceutical composition disclosed herein can optionally include,without limitation, other pharmaceutically acceptable components (orpharmaceutical components), including, without limitation, buffers,preservatives, tonicity adjusters, salts, antioxidants, osmolalityadjusting agents, physiological substances, pharmacological substances,bulking agents, emulsifying agents, wetting agents, sweetening orflavoring agents, and the like. Various buffers and means for adjustingpH can be used to prepare a pharmaceutical composition disclosed herein,provided that the resulting preparation is pharmaceutically acceptable.Such buffers include, without limitation, acetate buffers, boratebuffers, citrate buffers, phosphate buffers, neutral buffered saline,and phosphate buffered saline. It is understood that acids or bases canbe used to adjust the pH of a composition as needed. Pharmaceuticallyacceptable antioxidants include, without limitation, sodiummetabisulfite, sodium thiosulfate, acetylcysteine, butylatedhydroxyanisole, and butylated hydroxytoluene. Useful preservativesinclude, without limitation, benzalkonium chloride, chlorobutanol,thimerosal, phenylmercuric acetate, phenylmercuric nitrate, a stabilizedoxy chloro composition, such as, e.g., sodium chlorite and chelants,such as, e.g., DTPA or DTPA-bisamide, calcium DTPA, andCaNaDTPA-bisamide. Tonicity adjustors useful in a pharmaceuticalcomposition include, without limitation, salts such as, e.g., sodiumchloride, potassium chloride, mannitol or glycerin and otherpharmaceutically acceptable tonicity adjustor. The pharmaceuticalcomposition may be provided as a salt and can be formed with many acids,including but not limited to, hydrochloric, sulfuric, acetic, lactic,tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueousor other protonic solvents than are the corresponding free base forms.It is understood that these and other substances known in the art ofpharmacology can be included in a pharmaceutical composition useful inthe invention.

The combination of a RXR agonist and a thyroid hormone may also beincorporated into a drug delivery platform in order to achieve acontrolled release profile over time. Such a drug delivery platformcomprises the combination disclosed herein dispersed within a polymermatrix, typically a biodegradable, bioerodible, and/or bioresorbablepolymer matrix. As used herein, the term “polymer” refers to synthetichomo- or copolymers, naturally occurring homo- or copolymers, as well assynthetic modifications or derivatives thereof having a linear, branchedor star structure. Copolymers can be arranged in any form, such as,e.g., random, block, segmented, tapered blocks, graft, or triblock.Polymers are generally condensation polymers. Polymers can be furthermodified to enhance their mechanical or degradation properties byintroducing cross-linking agents or changing the hydrophobicity of theside residues. If crosslinked, polymers are usually less than 5%crosslinked, usually less than 1% crosslinked.

Suitable polymers include, without limitation, alginates, aliphaticpolyesters, polyalkylene oxalates, polyamides, polyamidoesters,polyanhydrides, polycarbonates, polyesters, polyethylene glycol,polyhydroxyaliphatic carboxylic acids, polyorthoesters, polyoxaesters,polypeptides, polyphosphazenes, polysaccharides, and polyurethanes. Thepolymer usually comprises at least about 10% (w/w), at least about 20%(w/w), at least about 30% (w/w), at least about 40% (w/w), at leastabout 50% (w/w), at least about 60% (w/w), at least about 70% (w/w), atleast about 80% (w/w), or at least about 90% (w/w) of the drug deliveryplatform. Examples of biodegradable, bioerodible, and/or bioresorbablepolymers and methods useful to make a drug delivery platform aredescribed in, e.g., U.S. Pat. Nos. 4,756,911; 5,378,475; 7,048,946; U.S.Patent Publication 2005/0181017; U.S. Patent Publication 2005/0244464;U.S. Patent Publication 2011/0008437; each of which is incorporated byreference in its entirety.

In aspects of this embodiment, a polymer composing the matrix is apolypeptide such as, e.g., silk fibroin, keratin, or collagen. In otheraspects of this embodiment, a polymer composing the matrix is apolysaccharide such as, e.g., cellulose, agarose, elastin, chitosan,chitin, or a glycosaminoglycan like chondroitin sulfate, dermatansulfate, keratan sulfate, or hyaluronic acid. In yet other aspects ofthis embodiment, a polymer composing the matrix is a polyester such as,e.g., D-lactic acid, L-lactic acid, racemic lactic acid, glycolic acid,caprolactone, and combinations thereof.

One of ordinary skill in the art appreciates that the selection of asuitable polymer for forming a suitable disclosed drug delivery platformdepends on several factors. The more relevant factors in the selectionof the appropriate polymer(s), include, without limitation,compatibility of polymer with drug, desired release kinetics of drug,desired biodegradation kinetics of platform at implantation site,desired bioerodible kinetics of platform at implantation site, desiredbioresorbable kinetics of platform at implantation site, in vivomechanical performance of platform, processing temperatures,biocompatibility of platform, and patient tolerance. Other relevantfactors that, to some extent, dictate the in vitro and in vivo behaviorof the polymer include the chemical composition, spatial distribution ofthe constituents, the molecular weight of the polymer and the degree ofcrystallinity.

A drug delivery platform includes both a sustained release drug deliveryplatform and an extended release drug delivery platform. As used herein,the term “sustained release” refers to the release of a compound orcombination disclosed herein over a period of about seven days or more.As used herein, the term “extended release” refers to the release of acompound or combination disclosed herein over a period of time of lessthan about seven days.

In aspects of this embodiment, a sustained release drug deliveryplatform releases a a combination of a RXR agonist and a thyroid hormonewith substantially first order release kinetics over a period of, e.g.,about 7 days after administration, about 15 days after administration,about 30 days after administration, about 45 days after administration,about 60 days after administration, about 75 days after administration,or about 90 days after administration. In other aspects of thisembodiment, a sustained release drug delivery platform releases acombination disclosed herein with substantially first order releasekinetics over a period of, e.g., at least 7 days after administration,at least 15 days after administration, at least 30 days afteradministration, at least 45 days after administration, at least 60 daysafter administration, at least 75 days after administration, or at least90 days after administration.

In aspects of this embodiment, a drug delivery platform releases acombination disclosed herein with substantially first order releasekinetics over a period of, e.g., about 1 day after administration, about2 days after administration, about 3 days after administration, about 4days after administration, about 5 days after administration, or about 6days after administration. In other aspects of this embodiment, a drugdelivery platform releases a combination disclosed herein withsubstantially first order release kinetics over a period of, e.g., atmost 1 day after administration, at most 2 days after administration, atmost 3 days after administration, at most 4 days after administration,at most 5 days after administration, or at most 6 days afteradministration.

Aspects of the present disclosure include, in part, administering acombination of a RXR agonist and a thyroid hormone. As used herein, theterm “administering” means any delivery mechanism that provides acompound or a combination disclosed herein to an individual thatpotentially results in a clinically, therapeutically, or experimentallybeneficial result.

Administration of a combination disclosed herein include a variety ofenteral or parenteral approaches including, without limitation, oraladministration in any acceptable form, such as, e.g., tablet, liquid,capsule, powder, or the like; topical administration in any acceptableform, such as, e.g., drops, spray, creams, gels or ointments; buccal,nasal, and/or inhalation administration in any acceptable form; rectaladministration in any acceptable form; vaginal administration in anyacceptable form; intravascular administration in any acceptable form,such as, e.g., intravenous bolus injection, intravenous infusion,intra-arterial bolus injection, intra-arterial infusion and catheterinstillation into the vasculature; peri- and intra-tissue administrationin any acceptable form, such as, e.g., intraperitoneal injection,intramuscular injection, subcutaneous injection, subcutaneous infusion,intraocular injection, retinal injection, or sub-retinal injection orepidural injection; intravesicular administration in any acceptableform, such as, e.g., catheter instillation; and by placement device,such as, e.g., an implant, a stent, a patch, a pellet, a catheter, anosmotic pump, a suppository, a bioerodible delivery system, anon-bioerodible delivery system or another implanted extended or slowrelease system. An exemplary list of biodegradable polymers and methodsof use are described in, e.g., Handbook of Biodegradable Polymers(Abraham J. Domb et al., eds., Overseas Publishers Association, 1997).

A combination disclosed herein can be administered to a mammal using avariety of routes. Routes of administration suitable for treating ademyelination-related disorder as disclosed herein include both localand systemic administration. Local administration results insignificantly more delivery of a combination to a specific location ascompared to the entire body of the mammal, whereas, systemicadministration results in delivery of a combination to essentially theentire body of the individual. Routes of administration suitable for ortreating a nervous system disorder as disclosed herein also include bothcentral and peripheral administration. Central administration results indelivery of a combination to essentially the central nervous system ofthe individual and includes, e.g., nasal administration, intrathecaladministration, epidural administration as well as a cranial injectionor implant. Peripheral administration results in delivery of a compoundor a combination to essentially any area of an individual outside of thecentral nervous system and encompasses any route of administration otherthan direct administration to the spine or brain. The actual route ofadministration of a compound or a combination disclosed herein used canbe determined by a person of ordinary skill in the art by taking intoaccount factors, including, without limitation, the type of nervoussystem disorder, the location of the nervous system disorder, the causeof the nervous system disorder, the severity of the nervous systemdisorder, the duration of treatment desired, the degree of reliefdesired, the duration of relief desired, the particular compound orcombination used, the rate of excretion of the compound or combinationused, the pharmacodynamics of the compound or combination used, thenature of the other compounds to be included in the combination, theparticular route of administration, the particular characteristics,history and risk factors of the individual, such as, e.g., age, weight,general health and the like, the response of the individual to thetreatment, or any combination thereof. An effective dosage amount of acompound or a combination disclosed herein can thus readily bedetermined by the person of ordinary skill in the art considering allcriteria and utilizing his best judgment on the individual's behalf.

In an embodiment, a combination disclosed herein is administeredsystemically to a mammal. In another embodiment, a combination disclosedherein is administered locally to a mammal. In an aspect of thisembodiment, a combination disclosed herein is administered to a site ofa nervous system disorder of a mammal. In another aspect of thisembodiment, a combination disclosed herein is administered to the areaof a nervous system disorder of a mammal.

In other embodiments, the combination is administered directly to thenervous system by intrathecal administration, epidural administration,cranial injection or implant, or nasal administration.

In other embodiments, the RXR agonist is administered orally, buccally,by nasal, and/or inhalation administration, intravascularly,intravenously, by intraperitoneal injection, intramuscularly,subcutaneously, intraocularly injection, by epidural injection; or byintravesicular administration and the thyroid hormone is administeredorally. The RXR agonist and the thyroid hormone do not need to beadministered by the same route or on the same administration schedule.

Aspects of the present specification provide, in part, administering atherapeutically effective amount of a combination of a RXR agonist and athyroid hormone. As used herein, the term “therapeutically effectiveamount” is synonymous with “therapeutically effective dose” and whenused in reference to treating a nervous system disorder means theminimum dose of a combination necessary to achieve the desiredtherapeutic effect and includes a dose sufficient to reduce at least onesymptom associated with a nervous system disorder. In aspects of thisembodiment, a therapeutically effective amount of a combination reducesat least one symptom associated with a nervous system disorder by, e.g.,at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90% or at least 100%. Inother aspects of this embodiment, a therapeutically effective amount ofa compound or a combination disclosed herein reduces at least onesymptom associated with a nervous system disorder by, e.g., at most 10%,at most 20%, at most 30%, at most 40%, at most 50%, at most 60%, at most70%, at most 80%, at most 90% or at most 100%. In yet other aspects ofthis embodiment, a therapeutically effective amount of a compound or acombination disclosed herein reduces at least one symptom associatedwith a nervous system disorder by, e.g., about 10% to about 100%, about10% to about 90%, about 10% to about 80%, about 10% to about 70%, about10% to about 60%, about 10% to about 50%, about 10% to about 40%, about20% to about 100%, about 20% to about 90%, about 20% to about 80%, about20% to about 20%, about 20% to about 60%, about 20% to about 50%, about20% to about 40%, about 30% to about 100%, about 30% to about 90%, about30% to about 80%, about 30% to about 70%, about 30% to about 60%, orabout 30% to about 50%. In still other aspects of this embodiment, atherapeutically effective amount of a combination is the dosagesufficient to reduces at least one symptom associated with a nervoussystem disorder for, e.g., at least one week, at least one month, atleast two months, at least three months, at least four months, at leastfive months, at least six months, at least seven months, at least eightmonths, at least nine months, at least ten months, at least elevenmonths, or at least twelve months.

In further embodiments, treatment with the combination reduces at leastone symptom, at least two symptoms, at least three symptoms, at leastfour symptoms, or at least five symptoms of a nervous system disorder.

The amount of active component in a combination disclosed herein fortreating a nervous system disorder can be varied so that a suitabledosage is obtained. The actual therapeutically effective amount of acombination disclosed herein to be administered to a mammal can bedetermined by a person of ordinary skill in the art by taking intoaccount factors, including, without limitation, the type of thedemyelination-related disorder, the location of the nervous systemdisorder, the cause of the nervous system disorder, the severity of thenervous system disorder, the duration of treatment desired, the degreeof relief desired, the duration of relief desired, the particularcombination used, the rate of excretion of the combination, thepharmacodynamics of the combination, the nature of the other compoundsto be included in the combination, the particular route ofadministration, the particular characteristics, history and risk factorsof the individual, such as, e.g., age, weight, general health and thelike, the response of the individual to the treatment, or anycombination thereof. An effective dosage amount of a compound or acombination disclosed herein can thus readily be determined by theperson of ordinary skill in the art considering all criteria andutilizing his best judgment on the individual's behalf.

Additionally, where repeated administration of a combination disclosedherein is used, the actual effective amount of compound, composition, orcombination disclosed herein will further depend upon factors,including, without limitation, the frequency of administration, thehalf-life of the compound, composition, or combination disclosed herein,or any combination thereof. It is known by a person of ordinary skill inthe art that an effective amount of a compound or a combinationdisclosed herein can be extrapolated from in vitro assays and in vivoadministration studies using animal models prior to administration tohumans. Wde variations in the necessary effective amount are to beexpected in view of the differing efficiencies of the various routes ofadministration. For instance, oral administration generally would beexpected to require higher dosage levels than administration byintravenous or intravitreal injection. Variations in these dosage levelscan be adjusted using standard empirical routines of optimization, whichare well-known to a person of ordinary skill in the art. The precisetherapeutically effective dosage levels and patterns are preferablydetermined by the attending physician in consideration of theabove-identified factors.

As a non-limiting example, when administering a RXR agonist disclosedherein to a mammal, a therapeutically effective amount generally is inthe range of about 0.001 mg/kg/day to about 100.0 mg/kg/day. In aspectsof this embodiment, an effective amount of a compound disclosed hereincan be, e.g., about 0.01 mg/kg/day to about 0.1 mg/kg/day, about 0.03mg/kg/day to about 3.0 mg/kg/day, about 0.1 mg/kg/day to about 3.0mg/kg/day, or about 0.3 mg/kg/day to about 3.0 mg/kg/day. In yet otheraspects of this embodiment, a therapeutically effective amount of acompound disclosed herein can be, e.g., at least 0.001 mg/kg/day, atleast 0.01 mg/kg/day, at least 0.1 mg/kg/day, at least 1.0 mg/kg/day, atleast 10 mg/kg/day, or at least 100 mg/kg/day. In yet other aspects ofthis embodiment, a therapeutically effective amount of a compounddisclosed herein can be, e.g., at most 0.001 mg/kg/day, at most 0.01mg/kg/day, at most 0.1 mg/kg/day, at most 1.0 mg/kg/day, at most 10mg/kg/day, or at most 100 mg/kg/day.

In other embodiments, the RXR agonist is administered to a mammal in atherapeutically effective amount generally in the range of about 0.001mg/day to about 100 mg/day, about 0.1 mg/day to about 50 mg/day, about0.5 mg/day to about 40 mg/day, about 1 mg/day to about 30 mg/day, orabout 1 mg/day to about 20 mg/day.

Suitable thyroxine doses are generally from about 5 μg/day to about 250μg/day orally initially with an increase in dose every 2-4 weeks asneeded. In other embodiments, the suitable thyroxine dose is from about5 μg/day to about 225 μg/day, from about 7.5 μg/day to about 200 μg/day,from about 10 μg/day to about 175 μg/day, from about 12.5 μg/day toabout 150 μg/day, from about 15 μg/day to about 125 μg/day, from about17.5 μg/day to about 100 μg/day, from about 20 μg/day to about 100μg/day, from about 22.5 μg/day to about 100 μg/day, from about 25 μg/dayto about 100 μg/day, from about 5 μg/day to about 200 μg/day, from about5 μg/day to about 100 μg/day, from about 7.5 μg/day to about 90 μg/day,from about 10 μg/day to about 80 μg/day, from about 12.5 μg/day to about60 μg/day, or from about 15 μg/day to about 50 μg/day. Increases in doseare generally made in increments of about 5 μg/day, about 7.5 μg/day,about 10 μg/day, about 12.5 μg/day, about 15 μg/day, about 20 μg/day, orabout 25 μg/day. In certain embodiments, the suitable thyroid hormonedose is a dose able to produce serum levels of T4 in the top 50%, thetop 60%, the top 70%, the top 80%, or the top 90% of the normal rangefor the testing laboratory. As the normal range of T4 levels may vary bytesting laboratory, the target T4 levels are based on normal rangesdetermined for each particular testing laboratory.

Dosing can be single dosage or cumulative (serial dosing), and can bereadily determined by one skilled in the art. For instance, treatment ofa nervous system disorder may comprise a one-time administration of aneffective dose of a combination disclosed herein. As a non-limitingexample, an effective dose of a combination disclosed herein can beadministered once to a mammal, e.g., as a single injection or depositionat or near the site exhibiting a symptom of a nervous system disorder ora single oral administration of the combination. Alternatively,treatment of a nervous system disorder may comprise multipleadministrations of an effective dose of a combination disclosed hereincarried out over a range of time periods, such as, e.g., daily, onceevery few days, weekly, monthly or yearly. As a non-limiting example, acombination disclosed herein can be administered once or twice weekly toa mammal. The timing of administration can vary from mammal to mammal,depending upon such factors as the severity of a mammal's symptoms. Forexample, an effective dose of a combination disclosed herein can beadministered to a mammal once a month for an indefinite period of time,or until the mammal no longer requires therapy. A person of ordinaryskill in the art will recognize that the condition of the mammal can bemonitored throughout the course of treatment and that the effectiveamount of a combination disclosed herein that is administered can beadjusted accordingly. Additionally, each element of the combination, forexample the RXR agonist and the thyroid hormone, can be administered bydifferent routes and on different schedules and are optionallyadministered individually, although both compositions (the RXR agonistand the thyroid hormone) are administered to the individual such thatplasma levels of both compounds are detectable at the same time.

A combination disclosed herein as disclosed herein can also beadministered to a mammal in combination with other therapeutic compoundsto increase the overall therapeutic effect of the treatment. The use ofmultiple compounds to treat an indication can increase the beneficialeffects while reducing the presence of side effects.

Also disclosed herein are combinations of a RXR agonist and a thyroidhormone co-administered with one or more neurotrophic factors, includingbut not limited to brain-derived neurotrophic factor (BDNF),glial-derived neurotrophic factor (GDNF), nerve growth factor (NGF),neurotrophin-3 (NT-3), fibroblast growth factor, basic (bFGF), ciliaryneurotrophic factor (CNTF), neurotrophic factors-4/5 (NT-4/5),insulin-like growth factor (IGF), insulin; or another neurotrophicfactor; or a synthetic mimetic molecule effecting similar biologicalactivities as BDNF, GDNF, NGF, NT-3, bFGF, CNTF, NT-4/5, IGF, insulin oranother neurotrophic factor.

Administration of a combination of a RXR agonist and thyroid hormonewith a neurotrophic factor, or a neurotrophic factor mimetic, may beused to affect neuroprotection, i.e enhanced survival of various typesof neural system cells (including neurons and glial cells).

In addition, administration of a combination of a RXR agonist andthyroid hormone with a neurotrophic factor, or a neurotrophic factormimetic, may be used to effect repair of damaged neural system cells(including neurons and glial cells), as manifested by promotion ofneurite outgrowth, resulting in formation and/or restoration of neuralconnections; or formation or restoration of glial structures, such asmyelin sheaths around neurons, which are essential for supportingoptimal neuronal signal transmission and nervous system functions.

Specific examples of uses of a combination of a RXR agonist and thyroidhormone with a neurotrophic factor or neurotrophic factor mimeticinclude, but are not limited to: co-administration of a combination of athyroid hormone and a RXR agonist such as IRX4204 or bexarotene, withGDNF or a GDNF mimetic, to promote dopaminergic neuron survival, orpromote repair or restoration of dopaminergic neurons, in patients withParkinson's disease or other diseases of dopaminergic neurons;co-administration with GDNF or a GDNF mimetic to enhance survival orpromote repair or restoration of motor neurons in patients withamyotrophic lateral sclerosis; co-administration with BDNF or a BDNFmimetic, or with insulin or insulin-like growth factor, to enhancesurvival or promote repair or restoration of cortical or hippocampalneurons in Alzheimer's disease; or co-administration with NGF to enhancesurvival or promote repair or restoration of sensory neurons in patientswith peripheral neuropathies. Other combinations of a RXR agonist andthyroid hormone with other neurotrophic factors or neurotrophic factormimetics, may be used for enhancing survival or promoting repair orrestoration of neurons or glial cells for additional diseases of thecentral or peripheral nervous systems, including but not limited tomultiple sclerosis of various forms, including relapsing-remitting orprogressive multiple sclerosis; optic neuritis; stroke of variousetiologies; nervous system trauma of various types; neuropathies ofvarious etiologies; nervous system hypoxia; toxic insults of the nervoussystem of various types; dementias of various etiologies; retinopathiesof various etiologies; Huntington's disease, various synucleinopathiessuch as progressive supranuclear palsy; epilepsy; autism; schizophrenia;depression, or aging-related nervous system degeneration.

In the above embodiments, the neurotrophic factor or neurotrophic factormimetic may be delivered to the patient orally, or by a parenteralroute, or by a topical route such as nasally, or as an inhaledmedicament; or alternatively by means of an implantable or wearable slowrelease formulation or slow delivery device.

A combination of a RXR agonist and thyroid hormone and a neurotrophicfactor, or neurotrophic factor mimetic also may be used for in vitropromotion of survival or growth of neurons or glial cells of varioustypes, for subsequent implantation into the nervous system of a patientwith a neurologic disease.

Aspects of the present specification may also be described as follows:

EXAMPLES

The following non-limiting examples are provided for illustrativepurposes only in order to facilitate a more complete understanding ofrepresentative embodiments now contemplated. These examples should notbe construed to limit any of the embodiments described in the presentspecification, including those pertaining to the methods of treating anautoimmune disorder, a nervous disorder, and/or a demyelination-relateddisorder, using the RXR agonists in combination with a thyroid hormonedisclosed herein, uses of a RXR agonist and a thyroid hormone disclosedherein to manufacture a medicament and/or treat an autoimmune disorder,a nervous system disorder, and/or a demyelination-related disorder.

Example 1 Selective RXR Agonist, IRX4204, Exerts its Biological EffectsThrough RXR Signaling

To determine whether a RXR agonist can mediate its effects via RXRαreceptor homodimers, RXRβ receptor homodimers, RXRγ receptor homodimers,or any combination thereof, or the corresponding RAR/RXR heterodimers,receptor-mediated transactivation assays were performed. Fortransactivation assays assessing RXR homodimer signaling, CV-1 cellswere transfected with 1) an expression construct including a full lengthRXRα, RXRβ, or RXRγ; and 2) a rCRBPII/RXRE-tk-Luc reporter constructthat included RXR homodimer-specific RXRE/DR1 responsive element linkedto a luciferase gene. For transactivation assays assessing RAR/RXRheterodimer signaling, CV-1 cells were transfected with 1) an expressionconstruct comprising a fusion protein including an estrogen receptor(ER) DNA binding domain linked to the ligand binding domain of RARα,RARβ, or RARγ and 2) a ERE-tk-Luc reporter construct that included anestrogen receptor responsive element linked to a luciferase gene. TheER-RAR fusion proteins provided an accurate readout of only thetransfected ER-RAR. After transfection, CV-1 cells were treated with RXRagonist IRX4204 at increasing concentrations for 20 hours beforemeasuring luciferase activity. Luciferase activity is expressed aspercent of maximal activity obtained using 1 μM RXR agonist IRX4204 forRXRs and 1 μM all-trans-retinoic acid (ATRA) for RARs (Table 1). Dataare mean values±SE from five independent experiments.

TABLE 1 EC₅₀ (nM) EC₅₀ (nM) Efficacy (% of 1 μM IRX4204) Efficacy (% of1 μM ATRA) Compound RXRα RXRβ RXRγ RARα RARβ RARγ IRX4204 0.08 ± 0.010.47 ± 0.05 0.09 ± 0.01 >1,000 >1,000 >1,000 100 100 100

These results indicate that RXR agonist IRX4204 activated RXR receptorswith very high potency (EC₅₀<0.5 nM) for all three RXR subtypes (Table1). In contrast, EC₅₀ of the RXR agonist for RARs was >1,000 nM withminimal activity detected at 1 μM. This differencerepresents >2,000-fold selectivity for RXRs over RARs in functionaltransactivation assays. Additionally, these data demonstrate that RXRagonist IRX4204 was more than 1,000-fold more potent in activating RXRreceptors rather than RAR receptors. These results indicate that Tregdifferentiation was mediated through a RXR signaling pathway and not viaa RAR signaling pathway. Also, using appropriate receptor and reporterconstructs, RXR agonist IRX4204 was shown not to transactivate so called“permissive RXR heterodimers” PPAR/RXR, FXR/RXR and LXR/RXR (FIG. 1A-C).In this regard, RXR agonist IRX4204 is distinct from other RXR agonists.Additionally, IRX4204 selectively activates the Nurr1/RXR permissiveheterodimer (FIG. 1D). Thus, RXR agonist IRX4204 has a unique profile inthat it selectively activates only RXR homodimers and Nurr1/RXRheterodimers.

Example 2 Binding Affinity of RXR Agonists

To determine the binding affinity for a RXR agonist, competitivedisplacement assays were performed. RXRα, RXRβ, RXRγ, RARα, RARβ, orRARγ were expressed in SF21 cells using a baculovirus expression systemand the resulting proteins were purified. To determine the bindingaffinity for a RXR agonist for an RXR, purified RXRα, RXRβ, and RXRγwere separately incubated with 10 nM [³H]-9CRA, and the binding affinityof the RXR agonist IRX4204 was determined by competitive displacement of[³H]-9CRA from the receptor. To determine the binding affinity for a RXRagonist for an RAR, purified RARα, RARβ, and RARγ were incubated with 5nM [³H]-ATRA, and the binding affinity of the RXR agonist IRX4204 wasdetermined by competitive displacement of [³H]-ATRA from the receptor.Ki values are mean values of at least two independent experiments (Table2). Standard errors (±) among independent experiments are indicated.

As shown in Table 2, RXR agonist IRX4204 displayed high affinity forRXRα, RXRβ, and RXRγ with Ki values being 1.7, 16, and 43 nM,respectively. In contrast, the RXR agonist IRX4204 bound with very lowaffinity to each of the RARs (Ki values being >1,000 nM). These dataindicate that IRX4204 is highly selective for the RXRs relative to theRARs.

TABLE 2 RXR Binding Affinity RAR Binding Affinity Ki (nM) Ki (nM)Compound RXRα RXRβ RXRγ RARα RARβ RARγ IRX4204 1.7 ± 0.1 16 ± 1.0 43 ±3.0 6344 ± 674 7552 ± 638 4742 ± 405

Example 3 RXR Agonists Attenuate EAE in B6 Mice

To determine whether a RXR agonist can attenuate multiple sclerosis,C57BL/6 (B6) mice were immunized (day 0) to induce experimentalautoimmune encephalomyelitis (EAE) by subcutaneous (s.c.) injection atthe base of their spine with 200 μL of adjuvant containing 125 μg myelinoligodendrocyte glycoprotein peptide (35-55) (MOG peptide; PeptidesInternational, Louisville, Ky.) and 400 μg non-viable M. tuberculosisH37 desiccate emulsified in a mixture of incomplete Freund's adjuvantand phosphate buffered saline (PBS). Mice were also given 200 ng ofpertussis toxin in PBS administered by inter-peritoneal (i.p.) injectionon the same day as MOG emulsion injection (day 0) and 2 days later (day2). Starting on day 7 after immunization, mice were given the RXRagonist IRX4204 (50 μg), vehicle control (i.p.), thyroxine (T4), orIRX4204+thyroxine every other day for the duration of the experiment(n=6-7 mice/group). Statistics show the results of a Mann Whitney test(analyzed from start of treatment to the end of the experiment). Micewere scored using the following scale: 0—Mice have no disease, 1—Micehave distal limp tail or rear leg weakness (paresis), 1.5—Mice havedistal limp tail and rear leg weakness, 2—Mice have complete limp tailand rear leg weakness, 2.5—Mice have complete limp tail and weakness inboth rear legs, 3—Mice have complete limp tail and paralysis in bothrear legs, 3.5—Mice have complete limp tail, paralysis in both rearlegs, and forelimb weakness. Mice receiving a score of 3.5 wereimmediately euthanized.

FIG. 2 depicts scores of disease severity over time. The resultsindicate that administration of the RXR agonist IRX4204 at 50 μgsignificantly reduces the symptoms of EAE in mice. Efficacy of the RXRagonist was observed after the first administration (day 7) andmaintained throughout the course of the study (day 20). However, thecombination of IRX4204 and thyroxine reduced the symptoms of EAE in miceto an even greater degree (FIG. 2).

A dose titration experiment was also conducted in EAE mice. EAE wasinduced in 28 B6 mice with MOG/CFA and PT as above. Mice were scored onday 7 as indicated above and divided into groups by score so means areas equal as possible. Starting day 8, mice were scored and injected witha vehicle control or IRX4204 (50 μg, 100 μg, or 200 μg) every day.

The mice were weighed at the beginning of experiment and every day theyhad a score of 2.5 or higher and mice were euthanized if they lost 15%or more of their start weight. All mice that were treated with IRX4204had significantly less disease overall (FIG. 7). At the completion ofthe experiment, the vehicle control and 200 μg/day groups wereeuthanized and spleen and CNS samples obtained.

The spleen samples were evaluated for CD49d (FIG. 8A) and CCR6 (FIG.8B), and IRX4204 treatment lowered CCR6, but not CD49d, expression onCD4 T cells. Additionally, CD4+CD25hi cells (generally consisting ofTReg) were reduced, although the frequency was not altered (FIGS. 9A and9B). The total number of effector and memory CD4 T cells, as indicatedby CD44 expression, decreased with IRX4204 treatment (FIG. 12C) and thetotal number of recently activated CD4 T cells, as indicated byexpression of both CD69 and CD44, was also decreased with IRX4204treatment (FIG. 9D).

In the CNS, the total the total number of infiltrating CD4 T cells wasreduced with IRX4204 treatment (FIG. 10). Restimulation withPMA/lonomycin was used to help detect the cytokine production. Both IFNγ(FIGS. 11A and 11B) and TNF (FIGS. 11C and 11D) were significantlyreduced with treatment. Co-expression of IFNg and IL-17A by CD4 T cellsin CNS was quantified, but was not significantly different betweengroups (FIG. 12A-12C).

Example 4 RXR Agonist-Treated Mice have Reduced Central Nervous SystemInfiltrating Cells

To determine whether a RXR agonist can reduce central nervous system(CNS) infiltrating cells, C57BL/6 (B6) mice were treated as described inExample 6. On day 20 after immunization, mice were sacrificed andperfused with phosphate buffered saline (PBS). Brain and spinal cordtissue was isolated, digested with DNase and LIBERASE DL® (RocheDiagnostics, Indianapolis, Ind.) for 30 minutes, and homogenized through70 micron nylon mesh filters. Resulting cells were placed over a Percollgradient to remove myelin. The remaining cells (microglia and CNSinfiltrating cells) were counted, stained for molecules of interest, andrun on a flow cytometer. Based on the frequencies obtained by FACS ofthese cell populations, total cell numbers of CNS infiltratingleukocytes expressing CD45, including CD4⁺ T cells and CD11c⁺ CD11b⁺myeloid dendritic cells (DC), were calculated.

FIG. 3 depicts the number of CD4⁺ cells (FIG. 3A) or CD11c⁺ CD11b⁺ cells(myeloid DC; FIG. 3B) in mice treated with the RXR agonist IRX4204versus the vehicle control. There was a significant reduction in theinfiltration of both CD4⁺ cells and CD11c⁺ CD11b⁺ cells in to the CNS inanimals treated with a RXR agonist as compared to the control. It isexpected that if the mice were treated with a combination of IRX4204 andthyroxine, there would be a further reduction of infiltration of thesecells in to the CNS. As disease is propagated in the CNS through theCD4⁺ cells infiltrating the CNS and becoming re-activated by CD11c⁺CD11b⁺ cells, this suggests that part of the mechanism of action in thismodel is to limit the presence of the cells in the CNS.

Example 5 RXR Agonists Attenuate EAE in SJL Mice

To determine whether a RXR agonist can attenuate multiple sclerosis, SJLmice were immunized to induce EAE by s.c. injection at the base of theirspine with 200 μL of adjuvant containing 200 μg proteolipid proteins(139-151) (PLP peptide; Peptides International, Louisville, Ky.) and 400μg of non-viable M. tuberculosis H37 desiccate emulsified in a mixtureof incomplete Freund's adjuvant and PBS. Mice were also given 150 ng ofpertussis toxin in PBS i.p. on the same day as PLP emulsion injectionand 2 days later. Starting day 7 after immunization, mice were given theRXR agonist IRX4204 (50 μg) or vehicle control i.p. every other day forthe duration of the experiment (n=6 mice/group). Mice were scored usingthe scale described in Example 3.

The results indicate that administration of the RXR agonist IRX4204significantly reduces the symptoms of EAE in mice. Table 3 shows thefeatures of a RXR agonist IRX4204 treatment in SLJ mice. FIG. 4 depictsscores of disease severity over time. Efficacy of the RXR agonist wasobserved after the second administration (day 8) and maintainedthroughout the course of the study (day 14). It is expected that ifadministration of IRX4204 was combined with thyroxine treatment, therewould be a further reduction in the symptoms of EAE and disease severityscores.

TABLE 3 RXR agonist Treatment in SJL Mice Clinical Features VehicleIRX4204 Mean Maximum Score 3.2 ± 0.6 1.5 ± 1.4 Disease Incidence 6/6 4/6Death from Disease 4/6 0/6

Example 6 RXR Agonist IRX4204 as a Selective Activator of Nurr1/RXRPermissive Heterodimer

In order to determine which permissive RXR heterodimer is activated bythe RXR agonist IRX4204, receptor transactivation assays were carriedout as follows for PPARγ/RXR, FXR/RXR, LXRα/RXR, LXRβ/RXR, andNurr1/RXR. For PPARγ: CV-1 cells were transfected with3×(rAOX/DR1)-tk-Luc reporter gene and an expression vector for PPARγ.For FXR:CV-1 cells were transfected with 3×(IBABP/IRI)-tk-Luc reportergene and vectors for FXR and RXRα. For LXR:CV-1 cells were transfectedwith 3×(PLTP/LXRE)-tk-Luc reporter gene with vectors for LXRα or LXRβ.For Nurr1: COS7 cells were transfected with 3×NBRE-tk-luc reporter geneand full length Nurr-1 with or without full-length RXRα plasmid. Cellswere then treated with vehicle or IRX4204 for 20 hr. Luciferase datawere normalized to co-transfected β-gal activity. Luciferase activitywas expressed as percent of maximal activity obtained using specificagonists. Rosiglitazone (PPARγ), GW4064 (FXR), T0901317 (LXR). The dataindicate that IRX4204 does not activate FXR/RXR (FIG. 5A), LXRα/RXR orLXRβ/RXR (FIG. 5B), or PPARγ/RXR (FIG. 5C). In contrast, IRX4204potently (EC₅₀<1 nm) activates the Nurr1/RXR heterodimer. These datacollectively indicate that IRX4204 is a unique RXR agonist in that itselectively activates the Nurr1/RXR heterodimer but not the PPARγ/RXR,FXR/RXR or LXR/RXR heterodimers.

Example 7 Effect of RXR Agonists on Oligodendrocyte Precursor CellDifferentiation

The goal of this study was to evaluate the effect of IRX4204 ondifferentiation of oligodendrocyte precursor cells (OPCs) intooligodendrocytes. OPCs were generated from a neurosphere culture ofE14.5 PLP-EGFP (on C57BL/6J background) mouse brains. The isolated OPCswere treated with IRX4204 and/or T3 to evaluate the expression of greenfluorescent protein (EGFP), which correlates with differentiation ofOPCs into oligodendrocytes. The EGFP expressing cells were quantifiedwith Cellomics Neuronal Profiling Algorithm. The positive (T3) controldemonstrated differentiation of OPCs as expected. The resultsdemonstrate that IRX4204 promotes OPC differentiation intooligodendrocytes as shown by the increase in the number of the EGFPpositive cells compared to negative control (DMSO). All testedconcentrations except the lowest concentration (1⁻⁶ μM) showed asignificant increase in OPC differentiation into oligodendrocytes (FIG.6). However, addition of T3 to the IRX4204-treated cultures induced evenhigher levels of EGFR+oligodendrocytes demonstrating the significantbenefit of the combination of IRX4204 and thyroid hormone.

The EGFP expressing cells in controls and all compounds were quantifiedwith Cellomics Neuronal Profiling Algorithm. The experiment wassuccessful as demonstrated by the significant increase in % EGFP cellsin positive control (T3; 8.5%) compared to the negative control (DMSO;2.3%). IRX4204 promotes OPC differentiation into oligodendrocytes asdemonstrated by the dose dependent increase in the number of the EGFPpositive cells compared to negative control (DMSO). IRX4204 did not showany differences in total cell number and pyknotic cells compared tocontrols. The results from this study demonstrate that IRX4204 promotesOPC differentiation. The data show a dose-dependent increase in thepercentage of EGFP cells compared to the negative control. These dateindicate that IRX4204 promotes the growth of myelin-forming cells incell culture.

Example 8 IRX4204 Enhances Central Nervous System (CNS) Remyelination inan In Vivo Model by Acting Directly on the Remyelination Process

A focal toxin (ethidium bromide) induced rat model of demyelination isused to ascertain the direct effects of IRX4204 on acute demyelinationindependent of the immunomudulatory effects of IRX4204. The experimentuses rats of relatively advanced age (1 year) since such rats undergoremyelination in a less efficient manner, thereby providing data thatare more relevant to the clinical treatment of human patients withmultiple sclerosis or other demyelination disorders.

Focal demyelination is induced in one year old rats (approximately 300 gin weight) by injecting stereotactically 5 μl of ethidium bromidesolution (0.01% vol/vol in saline) in a bilateral manner into the caudalcerebellar peduncles (CCP). Starting seven days after injection of theethidium bromide, the rats are treated by oral gavage for fourteen days(day 7 to day 21 post-ethidium bromide treatment) with 10 mg/kg/day ofIRX4204 (in DMSO and corn oil), or the same dose of oral IRX4204 plus 20ng/g of subcutaneous thyroxine, or vehicles (DMSO and corn oil plusthyroxine vehicle) for fourteen days. The rats are killed on day 24post-ethidium bromide treatment for analysis of remyelination byquantitative polymerase chain reaction (qPCR) and microscopy.

Analysis of the lesions revealed the following: the densities of Olig2⁺oligodendrocyte lineage cells and CC1+ differentiated oligodendrocytesincreased in IRX4204-treated animals relative to vehicle treated animalsand increased further in the IRX4204 plus thyroxine animals;Nkx2.2+oligodendrocyte precursor cells (OPCs) increased inIRX4204-treated lesions relative to vehicle treated lesions and werehighest in IRX4204 plus thyroxine treated lesions. Also, real-time qPCRanalysis of lesion samples show an increase in Mbp expression and anincrease in Pdgfra expression indicating higher levels of myelinregeneration in IRX4204-treated animals with highest levels of Mbp andPdgfra expression seen in IRX4204 plus thyroxine animals.Ultrastructural analyses of CCP lesions further demonstrate that IRX4204plus thyroxine treatment results in more remyelinated axons in animalsthan IRX4204 only treatment which in turn leads to more remyelinatedaxons than vehicle treatment. AG-ratio analysis (this ratio is that ofaxon diameter to myelinated axon) also shows that IRX4204-reated animalshave a lower G-ratio than vehicle treated animals and that this lowerratio is due to the formation of thicker remyelinated sheathssurrounding axons in IRX4204-treated animals. The G-ratio was furtherreduced in animals treated with the combination of IRX4204 andthyroxine. All these findings are consistent with an increase in CNSremyelination in IRX4204-treated animals and an optimal increase inIRX4204 plus thyroxine treated animals.

Example 9 IRX4204 in Combination with Thyroid Hormone AcceleratesRemyelination in the Cuprizone/Rapamycin Mouse Model of ToxicDemyelination

The cuprizone (bis-cyclohexanone oxaldihydrazone) model facilitatesreliable, reproducible and unequivocal analysis of myelin parameters inboth white and grey matter. The cuprizone model is a model for toxicdemyelination. In this model, young mice are fed with the copperchelator cuprizone, leading to oligodendrocyte death and a subsequentreversible demyelination. Cuprizone-fed mice with rapamycin, a drug thatblocks mTOR and spontaneous remyelination, allows for betterquantification of oligodendrocyte turnover. In the acute cuprizoneparadigm, male C57BL/6 mice at 6 to 9 weeks of age are fed a diet ofchow mixed with 0.2% cuprizone over the course of 6 weeks. By the thirdweek of cuprizone feeding, consistent demyelination can be observed inthe corpus callosum, the largest white matter tract in the mouse brain.Demyelination reaches a maximum at 5 or 6 weeks. Chronic demyelinationcan be induced if C57BL/6 mice are maintained on a diet with cuprizonefor 12 weeks.

The goal of this study was to evaluate the remyelination potential ofIRX4204 in a mouse model of toxic demyelination. Previous studies havedemonstrated efficacy of IRX4204 in an EAE model of MS. Also, previousdata demonstrates that IRX4204 can induce significant oligodendrocyteprecursor cell (OPC) differentiation in vitro. The current study isconducted to further investigate the CNS effects of IRX4204 in acuprizone model of MS on remyelination and neuroprotection.

The animals (8 week-old male C57BL/6J mice) were subjected to cuprizonediet plus rapamycin injections (CR, 10 mg/kg) for 12 weeks to inducedemyelination in white matter (CC, corpus callosum). After 12 weeks, CRwas discontinued and subsets of animals were treated daily for 6 weekswith either vehicle (oral IRX4204 vehicle) or IRX4204 (10 mg/kg, PO).All animals were sacrificed after 12 weeks of CR or after further 6weeks of treatment to evaluate myelin in white matter (corpus callosum)and gray matter (hippocampus and cortex). In addition, the size ofmyelinated axons was quantified and the large myelinated axons werefurther assessed by 3D-electron microscopy (3D-EM).

Demyelinating diseases, such as MS, are characterized by myelin loss,chronic inflammation, and axonal and oligodendrocyte loss in the CNS.Although the etiology of MS remains unknown, the disease generallystarts with sporadic, acute episodes and develops over time into achronic and progressive state. The acute and chronic demyelinatedlesions of MS can be demonstrated in cuprizone-diet induced mouse modelsthat depend for severity upon the duration of cuprizone administration.Cuprizone induces extensive demyelination in adult mouse brain andsimultaneous administration of rapamycin blocks the differentiation ofoligodendrocytes and prevents spontaneous remyelination during thedemyelination phase. This model also demonstrates the hippocampaldemyelination in MS. When cuprizone+rapamycin (CR) is discontinued,there is quantifiable spontaneous remyelination in this model, which canbe modified by drug intervention in the remyelination process. The12-week CR model of demyelination provides an opportunity to evaluatethe therapeutic potential of new drugs to promote remyelination in themouse brain.

A total of 40 animals were included in the study, where all 40 animalsreceived CR demyelination for 12 weeks. After demyelination, a subset(n=10) of animals are sacrificed to serve as controls to assess baselinedemyelination. The remaining animals are divided into groups (n=15)which are treated daily with oral IRX4204 (10 mg/kg) or oral vehicle forIRX4204 for six weeks.

There was no significant difference in any of the groups with regard tobody weight.

Floating brain sections are immunostained with myelin prolipid protein(PLP) to visualize and quantify myelin in gray matter, hippocampus (FIG.16A) and cortex (FIG. 16B). The percentage area covered by PLP stainingin animals treated with vehicles only after discontinuation of thedemyelination regimen is significantly greater than in animals who weresacrificed immediately after CR demyelination demonstrating theoccurrence of spontaneous remyelination.

In this study, the 12-week demyelination model is used to assess CNSeffects of IRX4204, with and without thyroid hormone supplementation,following 6-weeks of treatment. The results from this study demonstratethat IRX4204 significantly increases the size of myelinated axons in thecorpus callosum (FIG. 17). In addition, these large myelinated fibersdemonstrate a healthy phenotype. Thus, IRX4204 and has a neuroprotectiveeffect on myelinated neurons.

Additionally, IRX4204 plus thyroxine increases the number and density ofmyelinated axons in white and gray matter in addition to increasing thesize of myelinated axons in the corpus callosum.

Example 10 Evaluation of the Neuroprotective Potential of IRX4204 andIRX4204+Thyroxine in a Mouse Model of Non-Immune Mediated Demyelination

The modified cuprizone model (cuprizone+rapamycin) facilitates reliable,reproducible and unequivocal analysis of neurodegeneration caused bydemyelination. SMI-32 immunostaining enables the visualization andquantification of swollen and transected axons (ovoids) in the corpuscallosum and enables the assessment of the extent of axonaldegeneration. There were four groups of mice in the study:cuprizone+rapamycin (CR) only (n=6), CR+vehicles (n=12), CR+IRX4204(n=12), and CR+IRX4204+thyroxine (n=12). The test articles wereadministered concurrently with CR for 6 weeks. IRX4204 was administeredorally once daily at 10 mg/kg body weight. Thyroxine (T4) treatment wasinitiated one day after initiation of the IRX4204 treatment. T4 wasadministered subcutaneously (SC) once daily at 20 ng/g body weight. TheCR+vehicles group received the IRX4204 vehicle (oral) and the T4 vehicle(SC). All animals were subjected to terminal blood collection todetermine plasma T4 levels. After sacrifice, the density of SMI-32positive ovoids per unit area was determined for each group. The higherthe SMI-32 positive ovoid density, the greater the extent of axonaldegeneration. There was a 13.3% reduction in SMI-32+ovoids in theIRX4204 group relative to the vehicles group indicating someneuroprotection by IRX4204 alone. However, the IRX4204+thyroxine groupgave a 37.5% reduction relative to the vehicles group indicating thatthe IRX4204 plus thyroxine combination provides a substantial degree ofneuroprotection from the CR induced neurotoxicity.

Example 11 A Human Clinical Trial to Demonstrate Effects of IRX4204 inParkinson's Disease

An open-label, single site clinical study of early Parkinson's Diseasesubjects treated with IRX4204 was conducted to determine whether thepreclinical promise of IRX4204 as a disease modifying agent for PD willtranslate to the clinical setting upon treatment of early PD patientswith IRX4204 as determined by Unified Parkinson's Disease Rating Scale(UPDRS) measurements and safety assessments. The changes in UPDRS scoreswere correlated with circulating thyroxine levels.

The objectives of this study were to further characterize the safety andtolerability of IRX4204 in early patients, particularly reduction in T4levels, and to evaluate the effect of treatment with IRX4204 on themotor symptoms of PD measured by the UPDRS.

The study endpoints were (1) the change in motor testing scores from endof dosing period (Day 17), and (2) changes in T4 levels.

(a) Design Overview

This was a single site, open-label study designed to examine efficacy(reduction in UPDRS scores) and safety of 3 dose levels of IRX4204 incohorts of early PD patients for a period of approximately two weeks. Inthe three cohorts, each subject reported to the clinical research siteon at least 3 occasions:

-   -   Screening (Visit 1)—Screening to determine eligibility (up to 30        days prior to Baseline Visit)    -   Baseline Period (Visit 2)—Treatment with IRX4204 began on Day 1.    -   Week 2 (Visit 3)—subjects returned to the clinic approximately        17 days after initiation of IRX4204 for safety and efficacy        evaluations.

Safety and tolerability was assessed through all study visits includingblood and urine samples for laboratory tests, ECGs, physicalexamination, neurological examination and assessments for adverseevents.

To qualify for study participation, subjects were required to meet thefollowing criteria: 40-80 years of age, inclusive; have a clinicaldiagnosis of PD based on the UK Brain Bank Criteria; participant hasHoehn and Yahr stage <3; participant may be treated with PD symptomatictherapy on a stable dose for at least 30 days prior to the ScreeningVisit. Dose levels of PD symptomatic therapies will remain stablethrough the study; must be willing and able to provide informed consent;females must be of either non-child bearing potential or must be willingto avoid pregnancy by using medically accepted contraception for 4 weeksprior to and 4 weeks following the last dose of study medication.

Subjects who met any of the following criteria were not included in thestudy: has any form of Parkinsonism other than idiopathic PD; arecurrently experiencing motor fluctuations (end of dose wearing off ordyskinesia) reflective of later stages of PD; has evidence of dementiaor significant cognitive dysfunction; has clinically significantabnormal laboratory value and/or clinically significant unstable medicalor psychiatric illness; the subject has any disorder that may interferewith drug absorption, distribution, metabolism or excretion; the subjecthas evidence of clinically significant gastrointestinal, cardiovascular,hepatic, pulmonary, or other disorder or disease; pregnancy orbreastfeeding.

The clinical site prepared the study drug for administration bydispensing the correct dosage (20 mg/day, 10 mg/day or 5 mg/day) ofIRX4204 for each subject. On Day 1, subjects received their first doseof IRX4204. After Day 1, IRX4204 drug dosing occurred at home daily.Patients took their daily dose of study medication with foodapproximately the same time each day, preferably between 8 AM and 10 AM.On Day 1, subjects received a 15-day supply of IRX4204 for a once dailydose of 20 mg, 10 mg, or 5 mg. Five subjects were recruited for each ofthe three dose levels. All fifteen subjects completed 15 days of dosing.

All subjects (n=52 total, n=12-13 per dose level) completed 15 days ofdosing and returned to the clinic at the end of 2 weeks (day 15-17) forUPDRS score determination and safety assessments including determinationof plasma thyroxine (T4) levels. Percent changes in Total Motor scores,Total UPDRS scores and plasma T4 values were determined according to thefollowing:

${{Percent}\mspace{14mu} {Change}} = {\frac{{{Baseline}\mspace{14mu} {Value}} - {2\mspace{14mu} {Week}\mspace{14mu} {Value}}}{{Baseline}\mspace{14mu} {Value}} \times 100}$

The average percent changes in Total Motor and Total UPDRS scores forthe three dose levels are given in Table 4. A negative score indicatesan improvement in the disease as measured by the comprehensive UPDRSevaluation. The largest therapeutic response to IRX4204 treatment asmeasured by the Total Motor score (−31.4%) was obtained for the lowestdose of IRX4204 (5 mg/day). Surprisingly, there was less efficacy, asmeasured by the Total Motor sores, at each of the higher doses, 10mg/day (11.7%) and 20 mg/day (−14.5%). Similar results were obtainedwhen the Total UPDRS scores were considered. The best therapeuticresponse was obtained with the 5 mg/day cohort (−18.7%). Each of thehigher doses, 10 mg/day and 20 mg/day, were progressively lessefficacious with total UPDRS changes of −13.6% and 6.6%, respectively.

TABLE 4 Dose Total Motor Change Total UPDRS Change 20 mg/day −14.5%−6.6% 10 mg/day −11.7% −13.6%  5 mg/day −31.4% −18.7%

The average percent changes in plasma T4 levels for the three cohortsare given in Table 5. The relationship between dose level and percentagereduction in plasma thyroxine (T4) was direct: the higher the dose ofIRX4204 the greater the decrease in T4 levels. The 20 mg/day dose ofIRX4204 leads to an almost complete abrogation of plasma T4 (98.8%reduction). Interestingly, this high dose of IRX4204 is associated withthe least efficacy (only a 6.6% reduction in Total UPDRS scores).

TABLE 5 Dose Change in TSH 20 mg/day −98.8% 10 mg/day −36.6%  5 mg/day−28.9%

These data in a human clinical trial clearly indicate that the reductionin thyroid hormone levels upon dosing with IRX4204 negatively impactsthe therapeutic benefit of IRX4204. The clinical trial data from showsan inverse relationship between suppression of the thyroid axis(manifested by suppression of TSH, thyroid stimulating hormone) andclinical improvement from baseline in total motor scores and UPDRS.

Example 12 A Human Clinical Trial to Demonstrate Effects of theCombination of IRX4204 and Thyroxine on Myelin Repair in MultipleSclerosis Patients with Relapsing-Remitting Disease

A double blinded, placebo-controlled proof of concept clinical trial ofthe combination of IRX4204 and thyroxine is conducted in multiplesclerosis (MS) patients to demonstrate the direct effects of IRX4204 onmyelin repair in patients with relapsing-remitting MS. Patients withrelapsing-remitting MS are recruited to participate in the clinicaltrial and are provided informed consent describing risks and potentialbenefits of participation. The MS patients are treated with one ofseveral dose levels of IRX4204, ranging from 1 mg/day to 40 mg/day, suchas 5 mg/day administered orally as capsules, once per day and thyroxine,administered at 12.5 μg/day to 250 μg/day orally, such as 50 μg/day.Some patients are randomized to receive a placebo dose using matchingcapsules, which do not contain IRX4204 or thyroxine. Patients are dosedfor a minimum of 30 days, and as long as 180 days. Patients are assessedfor the status of myelin damage and speed of repair of demyelination inMS lesions that occur over this period of time in their brains, spinalcords, and/or optic nerves. Quantitation of myelin damage and repair isperformed at baseline and periodically through the dosing, usingspecialized imaging methods, which specifically examine and quantitatemyelin damage and repair in these parts of the nervous system. Suchmethods include, but are not limited to, Positron Emission Tomography(PET) scanning, utilizing imaging agents such as the thioflavine-Tderivative 2-(4′-methylaminophenyl)-6-hydroxybenzothiazole (PIB), whichalso binds to amyloid plaques. This compound is useful for useful forquantitating myelin repair. Alternatively, magnetic resonance imaging(MRI) using special contrast agents that bind to or enhance theappearance of areas of myelin damage or repair is utilized; or specialMRI analytical algorithms, such as magnetization transfer imaging, ordiffusion tensor imaging, are utilized to quantitate myelin damage andrepair in the IRX4204 and thyroxine-treated patients compared to theplacebo-treated patients. Dose response relationships of theIRX4204/thyroxine combination to myelin protection or repair areanalyzed across the cohorts of patients treated with various dose levelsof IRX4204 and thyroxine. In addition to the quantitation of myelindamage and repair by imaging methods, the clinical status of the MSpatients' disease progression is preliminarily evaluated using standardclinical endpoints for MS clinical trials, such as the ExpandedDisability Status Scale (EDSS). The EDSS is a 10 point scale whichquantitates the MS patients' levels of disability by evaluating physicalactivities of daily life, such as walking, swallowing, bowel and bladderfunction, etc. In addition, visual acuity testing is performed toquantitate effects of the IRX4204/thyroxine combination on myelin damageand repair in the optic nerves. Substantial clinical benefit as measuredby one or more of the techniques described above is observed in groupstreated with a combination of IRX4204 and thyroxine compared to thevehicles combination group. Positive but less substantial clinicalbenefit is obtained in groups treated with a combination of IRX4204 plusthyroxine vehicle.

Example 13 A Human Clinical Trial to Demonstrate the Effects of aCombination of IRX4204 and Thyroxine Treatment on Progression ofDisability in Multiple Sclerosis Patients with Relapsing-RemittingDisease

A double blind, placebo-controlled, clinical trial to demonstrateevidence of benefit of IRX4204/thyroxine treatment on progression ofdisability in MS is conducted in MS patients with relapsing-remittingMS. Patients with relapsing-remitting MS are recruited to participate inthe clinical trial and are provided informed consent describing risksand potential befits of participation. The MS patients are randomized totreatment with a dose level of IRX4204, in the range of 1 to 40 mg/dayadministered orally, and a dose level of thyroxine (12.5 μg/day to 250μg/day orally) or matching placebo, for 24 months. The primary clinicalefficacy outcome measure is the EDSS, a 10 point scale which quantitatesthe MS patients' levels of disability by evaluating physical activitiesof daily life, such as walking, swallowing, bowel and bladder function,etc. The clinical trial uses a sample size selected to demonstrate to astatistically significant level, a difference in change in the mean EDSSover time, of a least 1 point, between the IRX4204/thyroxine-treatedgroup, and the placebos-treated group, at the end of 24 months oftreatment. In addition, in this clinical trial visual acuity testing isperformed to quantitate effects of IRX4204/thyroxine on myelin damageand repair in the optic nerves. A sample size is selected which willdemonstrate to a statistically significant level, a difference in changein visual acuity over time, of a least 1 line on the standard visualacuity chart, between the IRX4204/thyroxine-treated group, and theplacebo-treated group, at the end of 24 months of treatment.

Example 14 A Human Clinical Trial to Demonstrate the Effects of aCombination of IRX4204 and Thyroxine Treatment on Clinical Improvementin Parkinson's Disease

A double blind, placebo-controlled, clinical trial to demonstrateevidence of benefit of IRX4204/thyroxine treatment on progression ofdisability in Parkinson's disease (PD) is conducted in patients who haveprovided informed consent describing risks and potential befits ofparticipation. The PD patients are randomized to treatment with a doselevel of IRX4204, in the range of 1 to 40 mg/day administered orally,and a dose level of thyroxine (12.5 μg/day to 250 μg/day orally) ormatching placebo, for 24 months. The primary clinical efficacy outcomemeasure is the Unified Parkinson's Disease Rating Scale (UPDRS). TheUPDRS is a rating tool to follow the longitudinal course of PD. It ismade up of the 1) mentation, behavior, and mood, 2) activities of dailyliving (ADL) and 3) motor sections. These are evaluated by interview.Some sections require multiple grades assigned to each extremity. Atotal of 199 points are possible with 199 representing the worst (total)disability) and 0 indicating no disability.

The clinical trial uses a sample size selected to demonstrate to astatistically significant level, a difference in change in the meanUPDRS over time, between the IRX4204/thyroxine-treated group, and theplacebo-treated group, at the end of 24 months of treatment.

Example 15 A Human Clinical Trial to Demonstrate the Effects of aCombination of IRX4204 and Thyroxine Treatment on Clinical Improvementin Alzheimer's Disease

A double blind, placebo-controlled, clinical trial to demonstrateevidence of benefit of IRX4204/thyroxine treatment on progression ofcognitive impairment in Alzheimer's disease (AD) is conducted inpatients who have provided informed consent describing risks andpotential befits of participation. The AD patients are randomized totreatment with a dose level of IRX4204, in the range of 1 to 40 mg/dayadministered orally, and a dose level of thyroxine (12.5 μg/day to 250μg/day orally) or matching placebo, for 24 months. The primary clinicalefficacy outcome measure is the Mini-Mental State Examination (MMSE),and optionally includes one or more of the Functional AssessmentQuestionnaire (FAQ), Physical Self-Maintenance Scale (PSMS), andNeuropsychiatric Inventory (NPI). The clinical trial uses a sample sizeselected to demonstrate to a statistically significant level, adifference in change in the mean MMSE over time, between theIRX4204/thyroxine-treated group, and the placebo-treated group, at theend of 24 months of treatment.

Example 16 Effect of IRX4204 in Parkinson's Disease Model

The purpose of this study was to evaluate IRX4204 treatment foramelioration of behavioral deficits in the rat 6-OHDA induced ParkinsonDisease (PD) model. The rat model of PD was produced by unilateral intrastriatum injection of the neurotoxin 6-hydroxydopamine (6-OHDA). Thisinjection produces dopaminergic (DA) neuron loss on the injected sidewhile sparing the contralateral DA neurons. The study design is depictedin Table 6.

TABLE 6 Dose Level Dose Volume Group Group of Test Item of Test ItemDosing # Size Test Item Route (mg/kg) (ml/kg) Regimen Testing Regimen 1n = 13 Vehicle TA1 PO NA 5 Once daily Paw Placement/ Vehicle TA2 SC 1from day 4 cylinder test: Day −1 2 n = 13 TA1 PO 10 5 until the(baseline), 3, 10, Vehicle TA2 SC NA 1 end of the 17, and 24. 3 n = 13Vehicle TA1 PO NA 5 study (day 24) TA2 SC T3: 1.5 μg/kg 1 T4: 9 μg/kg 4n = 12 TA1 PO 10 5 TA2 SC T3: 1.5 μg/kg 1 T4: 9 μg/kg

The paw placement (cylinder test) was used for assessment of the damage.This test assessed a rat's independent forelimb use to support the bodyagainst the walls of a cylindrical enclosure. The test took advantage ofthe animals' innate drive to explore a novel environment by standing onthe hind limbs and leaning towards the enclosing walls.

To perform this test, rats were placed individually in a glass cylinder(21 cm diameter, 34 cm height) and wall exploration was recorded for 3minutes. No habituation to the cylinder prior to recording was allowed.

The statistical analysis was performed as ratio between the intact andimpaired legs (R/L ratio). The ratio was expressed as the values ofintact right+both forelimbs divided by the values of impaired left+bothforelimbs. A lower value of the ratio means greater healing of the6-OHDA induced brain damage.

All treated animals gained weight throughout the study. The mean bodyweight of animals treated with the test item IRX4204 (TA1) with thevehicle of TA2 (group 2) or in combination with thyroxine andtriiodothyronine (TA2; group 4) were significantly higher than thevehicle treated group (Group 1) on study days 17 and 24 (157.17±2.93%for Group 2 and 157.61±3.54% for Group 4 vs. 142.62±2.93% for theVehicle group on day 24; p<0.05).

All animals with R/L ratio >1.5 were included in the study (ratiobetween the intact (R) and impaired legs (L) was expressed as the valuesof intact right+both forelimbs divided into the values of impairedleft+both forelimbs).

Paw placement was measured prior to induction of lesion (baseline) andagain 3 days after 6-OHDA injection, which was one day prior to IRX4204treatment. Once a week during three weeks (study days 10, 17 and 24),the animals were re-tested for their performance in the paw placementtest.

Animals were pre-selected based on the R/L ratio on study day 3, whenthe averaged ratio between the injured side and the intact side wasincreased relative to baseline levels (1.01±0.01 prior to surgery vs.6.49±0.59, 3 days after surgery).

As shown in FIG. 13, treatment with IRX4204 (TA1) with the vehicle ofTA2 (group 2) or in combination with thyroxine and triiodothyronine(TA2; group 4) significantly reduced the mean calculated R/L ratio,compared to the vehicle treated group (group 1) on study day 10(2.76±0.57 for Group 2 and 2.86±0.76 for Group 4 vs. 6.33±1.41 for theVehicle group; p<0.05).

The mean calculated ratio was lower in these groups compared to thevehicle group also on study days 17 and 24, however this ratio was notstatistically significant.

The average value of the ratio was calculated from the four values fromdays 3, 10, 17 and 24. The calculated values for group 2 and group 4 are3.79 and 3.14, respectively. This indicates that group 4 (IRX4204 incombination with thyroxine and triiodothyronine) is more effective thangroup 2 (IRX4204) alone.

Example 17 Mouse Oligodendrocyte Progenitor Cell Differentiation in thePresence of Vitamin D

The purpose of this study was to assess possible effects of IRX4204 incombination with vitamin D, or vitamin D and triiodothyronine (T3), ondifferentiation of mouse oligodendrocyte progenitor cells (OPCs) intooligodendrocytes. OPCs were derived from plp-EGFP expressing mice.

Therapeutic agents were tested in 96-well plates (6 wells perconcentration). Negative and positive controls (DMSO or 10 ng/ml T3thyroid hormone) were included in each plate. All media contained 0.1%DMSO and 0.1% EtOH. At the end of the 5-day treatment, cells were imagedon Cellomics in two channels and algorithms were used to count nucleiand EGFP+oligodendrocytes.

Surprisingly, it was observed that different doses of vitamin D incombination with IRX4204 showed a negative effect in oligodendrocyteproduction (FIG. 14). The production of oligodendrocytes in response toa three regimen treatment (IRX4204, Vitamin D and T3) was slightlyhigher than that of the treatment without T3 (IRX4204 and Vitamin D).This suggests an additive effect of T3 in the three regimen combination.

Example 18 Mouse Oligodendrocyte Progenitor Cell Differentiation

The purpose of this study was to assess possible effects of IRX4204 incombination with triiodothyronine (T3), on differentiation of mouseoligodendrocyte progenitor cells (OPCs) into oligodendrocytes. OPCs werederived from plp-EGFP expressing mice.

Therapeutic agents were tested in 96-well plates (6 wells perconcentration). Negative and positive controls (DMSO or 10 ng/ml T3thyroid hormone) were included in each plate. All media contained 0.1%DMSO. At the end of the 5-day treatment, cells were imaged on Cellomicsin two channels and algorithms were used to count nuclei andEGFP+oligodendrocytes.

FIG. 15A-C show clear dose-responses in oligodendrocyte production inresponse to different doses of IRX4204 and T3. The production ofoligodendrocytes in response to combination treatments of IRX4204 and T3was more than that of individual treatment alone in all conditions. Thissuggests an additive, or potentially a synergistic, effect in drivingoligodendrocyte precursor cell differentiation between IRX4204 and T3.Similar results were obtained when cells were stained with MBP antibodyand quantified (data not shown). These data suggest that a combinationof IRX4204 and T3 (or T4) will be optimal in remyelination.

Example 19 Neuroprotective Effect of IRX4204 in a Mouse Model ofDemyelination

The goal of this study was to evaluate the neuroprotective effect ofIRX4204 in a mouse model of non-immune mediated demyelination.

In this study, the 6-week demyelination model was used to assessneuroprotective potential of IRX4204 following 6-week concurrenttreatment during demyelination. A sub-group of animals were treated withT4 along with IRX4204. The results from this study demonstrate thatIRX4204 promotes neuroprotection without reducing the extent ofdemyelination in the corpus callosum.

Animals (8 week-old male C57BL/6J mice) were subjected to cuprizone dietplus rapamycin injections (CR) for 6 weeks to induce demyelination.Animals were treated with either vehicle or IRX4204 (10 mg/kg, PO), orIRX4204+T4 (10 mg/kg, PO, and 20 ng/g, SQ) daily for the entire 6 weeksduring demyelination. All animals were sacrificed after 6 weeks of CR toevaluate axonal integrity and microglial/macrophage activity in thewhite matter (corpus callosum, CC). Two groups (Vehicle and IRX4204+T4)were further examined for any protective effects on the extent ofmyelination in the CC.

There was a significant reduction in axonal transection as shown by thedecrease in the number of SMI32 positive axonal ovoids in the animalstreated with IRX4204+T4. However, there was no difference inmicroglial/macrophage activation and the number of myelinated axons inthe CC between the Vehicle and IRX4204+T4 groups. These findings supporta neuroprotective role of IRX4204 mediated by a potential direct effecton demyelinated axons.

A total of 50 animals were included in the study, where 43 animalsreceived CR demyelination for 6 weeks. During demyelination, a subset(n=7) of animals were kept on normal diet to serve as naïve age-matchedcontrols. The remaining animals received IRX4204 (n=14) or vehicle(n=14) or IRX4204+T4 (n=15) for 6 weeks concurrently during CR. Therewas no mortality during the in-life phase. In addition, there were noobserved health concerns during the treatment phase. All animals werealert and demonstrated proper grooming behavior. ANOVA analysis withmultiple group comparison showed no significant difference in terminalbody weights between IRX4204 or vehicle groups.

To assess thyroid hormone levels, terminal blood draws were taken toquantify the levels of T4. Animals treated with IRX4204 alone showed anapproximate 50% decrease in T4 levels when compared to vehicle controlanimals. Exogenous treatment with T4 corrected the thyroid hormonelevels as shown by increase in T4 levels in IRX4204+T4 group.

The floating brain sections were immunostained with SMI-32 to visualizeand quantify axonal ovoids in the CC. Animals that were subjected to CRshowed significantly higher numbers of SMI32 stained axonal ovoids in CCcompared to naïve animals. There was a significant decrease in thenumber of axonal ovoids in animals treated with both IRX4204 and T4compared to Vehicle. IRX4204 alone showed a trend towards decreasednumber of axonal ovoids but was not statistically different from theVehicle.

The floating brain sections were immunostained with lba-1 to visualizeand quantify microglia/macrophages in CC. Animals subjected to CR andtreated with Vehicle had a robust increase in lba1 staining in CCcompared to naïve animals. There was no difference in the levels of lba1staining in IRX4204 or IRX4204+T4 treated animals compared to vehicle.

Semi-thin (1 μm) sections of Epon-embedded CC tissue from animals thatreceived CR and Vehicle or IRX4204+T4 were used to visualize andquantify the number and density of myelinated axons in the CC. Animalsthat received CR and vehicle demonstrated robust demyelination of theCC. There was no significant difference in the number and density ofmyelinated axons in IRX4204+T4 treated animals when compared to vehicle.

IRX4204 treatment alone without T4 showed a trend towards decrease inaxonal ovoids, but it was statistically not different from vehicle.However, when animals that received IRX4204 were supplemented withexogenous T4 there was a significant decrease in the number of axonalovoids compared to vehicle. This data along with our previous in vivofindings support a neuroprotective effect of IRX4204. While there was adecrease in axonal ovoids, there was no significant difference inmicroglial/macrophage activation and myelination in the corpus callosumin Vehicle and IRX4204+T4 groups.

The finding that IRX4204 demonstrated a neuroprotective effect only inthe group with supplemental T4 suggests an enhanced effect of thecombination therapy over IRX4204 alone.

Quantification of myelinated axons in the corpus callosum showspotential responders and non-responders. FIG. 20A-C shows a highcorrelation between the number of axonal ovoids and myelinated axons(i.e. the animals that had very few ovoids had very high number anddensity of myelinated axons in the corpus callosum).

In closing, it is to be understood that although aspects of the presentspecification are highlighted by referring to specific embodiments, oneskilled in the art will readily appreciate that these disclosedembodiments are only illustrative of the principles of the subjectmatter disclosed herein. Therefore, it should be understood that thedisclosed subject matter is in no way limited to a particularmethodology, protocol, and/or reagent, etc., described herein. As such,various modifications or changes to or alternative configurations of thedisclosed subject matter can be made in accordance with the teachingsherein without departing from the spirit of the present specification.Lastly, the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which is defined solely by the claims.Accordingly, the present invention is not limited to that precisely asshown and described.

Certain embodiments of the present invention are described herein,including the best mode known to the inventors for carrying out theinvention. Of course, variations on these described embodiments willbecome apparent to those of ordinary skill in the art upon reading theforegoing description. The inventor expects skilled artisans to employsuch variations as appropriate, and the inventors intend for the presentinvention to be practiced otherwise than specifically described herein.Accordingly, this invention includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedembodiments in all possible variations thereof is encompassed by theinvention unless otherwise indicated herein or otherwise clearlycontradicted by context.

Groupings of alternative embodiments, elements, or steps of the presentinvention are not to be construed as limitations. Each group member maybe referred to and claimed individually or in any combination with othergroup members disclosed herein. It is anticipated that one or moremembers of a group may be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is deemed to contain the group asmodified thus fulfilling the written description of all Markush groupsused in the appended claims.

Unless otherwise indicated, all numbers expressing a characteristic,item, quantity, parameter, property, term, and so forth used in thepresent specification and claims are to be understood as being modifiedin all instances by the term “about.” As used herein, the term “about”means that the characteristic, item, quantity, parameter, property, orterm so qualified encompasses a range of plus or minus ten percent aboveand below the value of the stated characteristic, item, quantity,parameter, property, or term. Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the specification andattached claims are approximations that may vary. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical indication shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and values setting forth the broad scope ofthe invention are approximations, the numerical ranges and values setforth in the specific examples are reported as precisely as possible.Any numerical range or value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Recitation of numerical ranges ofvalues herein is merely intended to serve as a shorthand method ofreferring individually to each separate numerical value falling withinthe range. Unless otherwise indicated herein, each individual value of anumerical range is incorporated into the present specification as if itwere individually recited herein.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the present invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. All methods described herein can be performed in any suitableorder unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein is intended merely to betterilluminate the present invention and does not pose a limitation on thescope of the invention otherwise claimed. No language in the presentspecification should be construed as indicating any non-claimed elementessential to the practice of the invention.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the present invention so claimed areinherently or expressly described and enabled herein.

All patents, patent publications, and other publications referenced andidentified in the present specification are individually and expresslyincorporated herein by reference in their entirety for the purpose ofdescribing and disclosing, for example, the compositions andmethodologies described in such publications that might be used inconnection with the present invention. These publications are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing in this regard should be construed as an admissionthat the inventors are not entitled to antedate such disclosure byvirtue of prior invention or for any other reason. All statements as tothe date or representation as to the contents of these documents isbased on the information available to the applicants and does notconstitute any admission as to the correctness of the dates or contentsof these documents.

1. A method of treating stroke, the method comprising administering to ahuman individual in need thereof a therapeutically effective amount of aRXR agonist and a thyroid hormone, wherein administration of thecombination of the RXR agonist and the thyroid hormone treats the strokein the individual, wherein the RXR agonist is3,7-dimethyl-6(S),7(S)-methano,7-[1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphth-7-yl]2(E),4(E)heptadienoic acid, and has the structure of formula III:

and wherein the combination of RXR agonist and thyroid hormone causes agreater improvement in the stroke than the RXR agonist or thyroidhormone alone.
 2. The method according to claim 1 wherein thecombination of RXR agonist and thyroid hormone treats stroke in theindividual by both promoting remyelination and neuroprotection ofneurons and modulating the individual's immune system.
 3. The methodaccording to claim 1, wherein the therapeutically effective amount ofthe RXR agonist is about 0.001 mg/day to about 100 mg/day.
 4. The methodaccording to claim 1, wherein the therapeutically effective amount ofthe RXR agonist is about 1 mg/day to about 20 mg/day.
 5. The methodaccording to claim 1, wherein the thyroid hormone is thyroxine.
 6. Themethod according to claim 5, wherein the dose of thyroxine is about 12.5μg/day to about 250 μg/day.
 7. The method according to claim 1, whereinthe RXR agonist is administered by nasal administration.
 8. The methodaccording to claim 7, wherein both the RXR agonist and thyroid hormoneare administered by nasal administration.
 9. The method according toclaim 1, wherein the RXR agonist is administered orally.
 10. The methodaccording to claim 1, wherein the RXR agonist and the thyroid hormoneare administered substantially simultaneously.
 11. The method accordingto claim 1, wherein the RXR agonist and thyroid hormone are administeredon different schedules.
 12. The method according to claim 1, wherein thethyroid hormone is administered orally or subcutaneously.
 13. The methodaccording to claim 1, wherein treatment with the combination of RXRagonist and thyroid hormone reduces at least one symptom of stroke,wherein the at least one symptom reduced is inflammation, fatigue,dizziness, headache, malaise, elevated fever and high body temperature,extreme sensitivity to cold in the hands and feet, weakness andstiffness in muscles and joints, weight changes, digestive orgastrointestinal problems, low or high blood pressure, irritability,anxiety, or depression, blurred or double vision, ataxia, clonus,dysarthria, fatigue, clumsiness, hand paralysis, hemiparesis, genitalanesthesia, incoordination, paresthesias, ocular paralysis, impairedmuscle coordination, weakness (muscle), loss of sensation, impairedvision, neurological symptoms, unsteady gait, spastic paraparesis,incontinence, hearing problems, or speech problems.
 14. The method ofclaim 1, further comprising administration of a neurotrophic factor, orneurotrophic factor mimetic.
 15. The method of claim 1, furthercomprising administration of a neurotrophic factor.
 16. The method ofclaim 14, wherein the neurotrophic factor is brain-derived neurotrophicfactor (BDNF), glial-derived neurotrophic factor (GDNF), nerve growthfactor (NGF), neurotrophin-3 (NT-3), fibroblast growth factor, basic(bFGF), ciliary neurotrophic factor (CNTF), neurotrophic factors-4/5(NT-4/5), insulin-like growth factor (IGF), or insulin, or a mimetic ofany one of these neurotrophic factors.
 17. The method of claim 16,wherein the neurotrophic factor is GDNF.
 18. The method of claim 16,wherein the neurotrophic factor is insulin or insulin-like growthfactor.
 19. The method of claim 16, wherein the neurotrophic factor isBDNF.
 20. The method claim 16, wherein the neurotrophic factor is NGF.21. The method according to claim 14, wherein the therapeuticallyeffective amount of the RXR agonist is about 0.001 mg/day to about 20mg/day.
 22. The method according to claim 14, wherein thetherapeutically effective amount of the RXR agonist is about 1 mg/day toabout 10 mg/day.
 23. The method according to claim 14, wherein the RXRagonist is administered directly to the central nervous system.
 24. Themethod according to claim 14, wherein the combination of RXR agonist andthyroid hormone is administered directly to the central nervous system.25. The method according to claim 14, wherein the combination of RXRagonist, thyroid hormone, and neurotrophic factor is administereddirectly to the central nervous system.
 26. The method according toclaim 14, wherein the neurotrophic factor is administered directly tothe central nervous system.
 27. The method according to claim 14,wherein further combination with the neurotrophic factor promotesremyelination or neuroprotection.
 28. The method according to claim 14,wherein the neurotrophic factor is administered by nasal administration.29. The method according to claim 14, wherein the combination of RXRagonist, thyroid hormone, and neurotrophic factor is administered bynasal administration.
 30. The method according to claim 14, wherein theneurotrophic factor is administered intravenously, subcutaneously, orintramuscularly.